Zeiss LSM 880 Operating Manual

Summary of LSM 880

  • Page 1

    Ls m 880 l s m 880 nl o o p e ra ti ng m a nu a l october 2014 zen 2 (black edition).

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    Introduction zeiss lsm 880 ii 000000-2071-464 10/2014 v_01 knowledge of this manual is required for the operation of the instrument. Would you therefore please make yourself familiar with the contents of this manual and pay special attention to hints concerning the safe operation of the instrument. ...

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    Introduction lsm 880 zeiss 10/2014 v_01 000000-2071-464 iii how to make best use of the lsm 880 and lsm 880 nlo operating instructions: a few symbols in these operating instructions will help you to recognize the nature and purpose of information immediately: the warning symbol warns against hazards...

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    Introduction zeiss lsm 880 iv 000000-2071-464 10/2014 v_01 contents 1 system operation in this section you will find the most important steps and procedures of the lsm menu structure. The step-by-step description how to get an image will be shown by typical application examples including the windows...

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    Important notes lsm 880 zeiss 10/2014 v_01 000000-2071-464 1-i important notes for chapter 1 • visual macro editor does not allow saving .Czi file format. • auto z correction function works with laser powers smaller than 0.5 % but does not display the changes even if enable test is active. • do not ...

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    Important notes zeiss lsm 880 1-ii 000000-2071-464 10/2014 v_01 • the functions crop, split, delete and copy used in the file browser menu requires some time for gui update. • when using the channel unmixing function for airyscan data, the histogram of the resulting image still refers to the airysca...

  • Page 7: Chapter 1

    Chapter 1 - system operation lsm 880 contents zeiss 10/2014 v_01 000000-2071-464 1 chapter 1 system operation contents page 1 purpose .............................................................................................................. 8 1.1 software............................................

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    Chapter 1 - system operation zeiss contents lsm 880 2 000000-2071-464 10/2014 v_01 4.5.3 help – about .................................................................................................................. 37 4.6 main toolbar ..................................................................

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    Chapter 1 - system operation lsm 880 contents zeiss 10/2014 v_01 000000-2071-464 3 5.2.9.1 defining channels and tracks ....................................................................................... 116 5.2.9.2 ratio channels ....................................................................

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    Chapter 1 - system operation zeiss contents lsm 880 4 000000-2071-464 10/2014 v_01 5.3.4 setup – fit ..................................................................................................................... 192 5.3.4.1 defining a new model ...................................................

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    Chapter 1 - system operation lsm 880 contents zeiss 10/2014 v_01 000000-2071-464 5 5.4.12 processing – hdr-imaging ............................................................................................ 321 5.4.13 processing – stitch ................................................................

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    Chapter 1 - system operation zeiss contents lsm 880 6 000000-2071-464 10/2014 v_01 5.5.5.3 system options – hardware ........................................................................................... 411 5.5.5.4 system options – image display ...................................................

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    Chapter 1 - system operation lsm 880 contents zeiss 10/2014 v_01 000000-2071-464 7 6.11 colocalization view ................................................................................................... 472 6.11.1 how a scatter diagram is generated .................................................

  • Page 14: Purpose

    Chapter 1 - system operation zeiss purpose lsm 880 8 000000-2071-464 10/2014 v_01 1 purpose this section describes the operation of the laser scanning microscope with the zen 2 software. When starting up and operating the microscope system, mind the operating instruction manuals for the axio imager....

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    Chapter 1 - system operation lsm 880 purpose zeiss 10/2014 v_01 000000-2071-464 9 1.3 backup system backup prior to the delivery of each system, a backup of the entire system computer is created in the factory. This backup image belongs to the system and may be re-installed in order to restore the o...

  • Page 16: Hardware Aspects

    Chapter 1 - system operation zeiss hardware aspects lsm 880 10 000000-2071-464 10/2014 v_01 2 hardware aspects 2.1 principle of laser scanning microscopy to yield information on their inner structure by conventional transmitted-light microscopy, specimens have to be very thin and translucent; otherw...

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    Chapter 1 - system operation lsm 880 hardware aspects zeiss 10/2014 v_01 000000-2071-464 11 2.2 principle of fluorescence correlation spectroscopy fluorescence correlation spectroscopy (fcs) analyzes the diffusion time of molecules and their differences if they have bound together. This is done by f...

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    Chapter 1 - system operation zeiss hardware aspects lsm 880 12 000000-2071-464 10/2014 v_01 2.3 principle of airyscanning the airyscan detector consists of 32 elements based on gaasp pmt technology with each element detecting the emission signal of a focal spot much smaller than the typical 1 airy v...

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    Chapter 1 - system operation lsm 880 hardware aspects zeiss 10/2014 v_01 000000-2071-464 13 2.4 optical diagram of the lsm 880 (schematic) as airyscan detector mbs main beam splitter dbs dichroic beam splitter ndd non-descanned detector ef emission filter pmt photomultiplier hal halogen lamp t-pmt t...

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    Chapter 1 - system operation zeiss hardware aspects lsm 880 14 000000-2071-464 10/2014 v_01 2.5 performance features of the lsm 880 optical and mechanical aspects the highly integrated system design makes for the shortest possible optical paths, top-grade optical precision and high stability. The ai...

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    Chapter 1 - system operation lsm 880 hardware aspects zeiss 10/2014 v_01 000000-2071-464 15 2.6 microscope equipment of the lsm 880 lsm 880 is attached to either the rear port or side port of the inverted microscope axio observer.Z1 or to the upright microscopes axio imager.Z2 or axio examiner.Z1. O...

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    Chapter 1 - system operation zeiss hardware aspects lsm 880 16 000000-2071-464 10/2014 v_01 (3) transmitted-light illumination - the illuminator support contains a security circuit which activates a shutter preventing laser light from reaching the stand when the support is moved to the back. A compl...

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    Chapter 1 - system operation lsm 880 hardware aspects zeiss 10/2014 v_01 000000-2071-464 17 2.7 computer hardware and software the lsm 880 (nlo) airyscan / big.2 are controlled via a standard high-end pentium / xeon pc. Linking to the electronic control system is made via gigabit ethernet interface....

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    Chapter 1 - system operation zeiss startup and shutdown of the system lsm 880 18 000000-2071-464 10/2014 v_01 3 startup and shutdown of the system 3.1 startup of the system the lsm system is equipped with a main power switch and two further switches labeled system/pc and components that are located ...

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    Chapter 1 - system operation lsm 880 startup and shutdown of the system zeiss 10/2014 v_01 000000-2071-464 19 system maintenance tool icon with a calibration objective and correct system configuration, the maintenance tool allows convenient self adjustment of the lsm 880 systems. The optical beam pa...

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    Chapter 1 - system operation zeiss startup and shutdown of the system lsm 880 20 000000-2071-464 10/2014 v_01 3.1.2 starting zen • to start the software double click the zen icon on your desktop! − during loading the program the zen access window will appear (fig. 6). It displays the zen symbol in t...

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    Chapter 1 - system operation lsm 880 startup and shutdown of the system zeiss 10/2014 v_01 000000-2071-464 21 the login zen dialog allows you to boot the software in different modes. If the boot status is closed, the options are start system and image processing . A third option, offline/demo , beco...

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    Chapter 1 - system operation zeiss startup and shutdown of the system lsm 880 22 000000-2071-464 10/2014 v_01 − if recent… is pressed the last couple of databases that were in use will be displayed (fig. 12). Click on the one you want to select. By pressing the choose… button windows explorer will o...

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    Chapter 1 - system operation lsm 880 startup and shutdown of the system zeiss 10/2014 v_01 000000-2071-464 23 3.2 shutdown procedure never shut down the computer by its main switch while the zen software is still active, or else you will lose the currently set operating parameters and the images jus...

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    Chapter 1 - system operation zeiss startup and shutdown of the system lsm 880 24 000000-2071-464 10/2014 v_01 the software can be shut down from file menu of the main toolbar by selecting exit (fig. 13) or by pressing the window close button (symbolized by a cross) of the zen window. If in maintain ...

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    Chapter 1 - system operation lsm 880 startup and shutdown of the system zeiss 10/2014 v_01 000000-2071-464 25 3.2.2 switching system power off after having exited the zen software, you may turn off the whole system with the following shutdown procedure. • make sure you have saved all your image data...

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    Chapter 1 - system operation zeiss introduction to the software application layout lsm 880 26 000000-2071-464 10/2014 v_01 4 introduction to the software application layout 4.1 overview on the screen layout fig. 15 zen main application window after startup with empty image container f ig. 16 zen mai...

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    Chapter 1 - system operation lsm 880 introduction to the software application layout zeiss 10/2014 v_01 000000-2071-464 27 4.2 introduction to zen zen - efficient navigation - is the new software for the lsm family from zeiss. With the launch of this software in 2007 zeiss sets new standards in appl...

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    Chapter 1 - system operation zeiss introduction to the software application layout lsm 880 28 000000-2071-464 10/2014 v_01 the show all concept ensures that tool panels are never more complex than needed. In the basic mode of the tools show all is deactivated and the tools show only the most relevan...

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    Chapter 1 - system operation lsm 880 introduction to the software application layout zeiss 10/2014 v_01 000000-2071-464 29 fig. 18 zen window layout configuration more features of zen include: − the user can add more columns to the left tool area or detach individual tools to position them anywhere ...

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    Chapter 1 - system operation zeiss introduction to the software application layout lsm 880 30 000000-2071-464 10/2014 v_01 4.3 function elements function element description / explanation tool group tool (closed) or tool window (opened) panel (e.G.: speed panel) − field with a subset of tools of a t...

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    Chapter 1 - system operation lsm 880 introduction to the software application layout zeiss 10/2014 v_01 000000-2071-464 31 slider with input ("spin") box and arrows − setting of numbers in the relevant input box by moving the slider or clicking on the arrow buttons or clicking on the slider and movi...

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    Chapter 1 - system operation zeiss introduction to the software application layout lsm 880 32 000000-2071-464 10/2014 v_01 4.4 application bar the application bar includes the following control elements of the zen software application window: starts the online help minimizes the application window s...

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    Chapter 1 - system operation lsm 880 introduction to the software application layout zeiss 10/2014 v_01 000000-2071-464 33 menu overview view text view displays the image tabs with text only. Small thumbnail view displays the image tabs with small thumbnails. Maintain set spline opens a dialog for s...

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    Chapter 1 - system operation zeiss introduction to the software application layout lsm 880 34 000000-2071-464 10/2014 v_01 for detailed information about the image acquisition functions see section acquisition tab . The functionalities of the macro menu are described in chapter 2: macros and visual ...

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    Chapter 1 - system operation lsm 880 introduction to the software application layout zeiss 10/2014 v_01 000000-2071-464 35 4.5.2 maintain the maintain menu from the menu bar contains additional modules to check and guarantee the interference-free operation of all the software and hardware components...

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    Chapter 1 - system operation zeiss introduction to the software application layout lsm 880 36 000000-2071-464 10/2014 v_01 4.5.2.2 camera this function allows the user to adjust the white balance and color balance of a connected camera. • click on camera in the maintain list. − the camera color adju...

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    Chapter 1 - system operation lsm 880 introduction to the software application layout zeiss 10/2014 v_01 000000-2071-464 37 4.5.3 help – about the about window can be accessed via the help item in the menu bar clicking about opens the following dialog: fig. 25 about window the panel hosts important i...

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    Chapter 1 - system operation zeiss introduction to the software application layout lsm 880 38 000000-2071-464 10/2014 v_01 4.6 main toolbar fig. 26 main toolbar, left side fig. 27 main toolbar, right side workspace configuration this function allows loading, saving or deleting a workspace configurat...

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    Chapter 1 - system operation lsm 880 introduction to the software application layout zeiss 10/2014 v_01 000000-2071-464 39 • to load a workspace configuration, click on the button. The following list box will appear: selecting on of the list entries will load the respective configuration. • to delet...

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    Chapter 1 - system operation zeiss introduction to the software application layout lsm 880 40 000000-2071-464 10/2014 v_01 4.7 status bar fig. 28 status bar progress bar shows an overview of all running processes. If only one process is running the details of this one are shown. If more than one pro...

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    Chapter 1 - system operation lsm 880 introduction to the software application layout zeiss 10/2014 v_01 000000-2071-464 41 4.8 left tool area main tool tabs switches between the locate , acquisition , processing and maintain main tools to operate the included tool action buttons action buttons are o...

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    Chapter 1 - system operation zeiss introduction to the software application layout lsm 880 42 000000-2071-464 10/2014 v_01 multidimensional acquisition: tools for all multidimensional imaging the tools of this tool group will only appear if selected in the selection panel below the start buttons. On...

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    Chapter 1 - system operation lsm 880 introduction to the software application layout zeiss 10/2014 v_01 000000-2071-464 43 4.8.1 tool groups and tools multiple columns layout of tool groups this function allows moving one or several tool groups in a second or third column of the left tool area . Thi...

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    Chapter 1 - system operation zeiss introduction to the software application layout lsm 880 44 000000-2071-464 10/2014 v_01 4.8.2 context menu of the left tool area there are 2 different context menus in the left tool area with different functionalities available. New acquisition document opens an em...

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    Chapter 1 - system operation lsm 880 introduction to the software application layout zeiss 10/2014 v_01 000000-2071-464 45 4.9 center screen area the center screen area is used for displaying scanned images or to show images in the available image views. Using the context menu of the center screen a...

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    Chapter 1 - system operation zeiss introduction to the software application layout lsm 880 46 000000-2071-464 10/2014 v_01 image tabs for each opened image one image tab is shown in the header of the actual container. Three modes can be selected using the context menu of the center screen area (see ...

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    Chapter 1 - system operation lsm 880 introduction to the software application layout zeiss 10/2014 v_01 000000-2071-464 47 arrow down button clicking on this button shows a list with all opened images for fast image selection. Using the drag and drop function, one or several images can be moved into...

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    Chapter 1 - system operation zeiss introduction to the software application layout lsm 880 48 000000-2071-464 10/2014 v_01 4.10 right tool area the right tool area is used for displaying and handling the opened images, e.G. Save or close. The view of this area can be varied individually, e.G. Expand...

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    Chapter 1 - system operation lsm 880 introduction to the software application layout zeiss 10/2014 v_01 000000-2071-464 49 big view button shows images with the textual characters and a big image preview. Save status icon this icon appears in the image that is not saved yet. Image information displa...

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    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 50 000000-2071-464 10/2014 v_01 5 left tool area and hardware control tools the left tool area contains all tools for system operation, image acquisition, image processing and maintenance. The major functions are or...

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    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 51 5.1 locate tab locate contains all functions of the microscope for visual inspection of the sample through the eyepieces (fig. 41). You can open the shutters for transmitted light (ha...

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    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 52 000000-2071-464 10/2014 v_01 to delete a configuration press the delete button ( ). This opens the delete configuration panel (fig. 43). Select the ocular configuration from the configurations list by highlightin...

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    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 53 these microscope functions can also be operated directly on the microscope stand via assigned buttons or through the touch screen, if available. In that case, any changes are recorded...

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    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 54 000000-2071-464 10/2014 v_01 hal transmitted light open/close shutter button: the shutter for transmitted light from the hal lamp is switched on and off by repeated pressing (fig. 49). Note that it is equivalent ...

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    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 55 focus button: if the focus button is pressed, the focus control panel will appear (fig. 52). The z-position display box shows the current z-position. The number can be edited by typin...

  • Page 62

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 56 000000-2071-464 10/2014 v_01 tube lens button: the tube lens can be selected via a graphical pop- up menu (fig. 55). Lens 1x is used for ocular observation and widefield imaging using an arc lamp, lens lsm is use...

  • Page 63

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 57 visual inspection of the cells via binoculars • in the main toolbar, activate the locate tab. • choose from the locate tab the microscope control tool. For transmitted-light observati...

  • Page 64

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 58 000000-2071-464 10/2014 v_01 (1) conventional setting of the microscope axio observer.Z1 the recording of microscope settings works as described for the microscope axio imager. For the conventional setting of the...

  • Page 65

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 59 (3) microscope settings on axio observer for reflected-light observation (epi-fluorescence) • turn on the hbo 100 power supply switch (fig. 59/ 1 ). • click on the reflected light but...

  • Page 66

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 60 000000-2071-464 10/2014 v_01 5.1.2 microscope control tool for axio imager.Z2 • click on locate tab in the left tool area. Click online if needed. • the currently set light path of the microscope is displayed. • ...

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    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 61 (1) microscope settings on axio imager for transmitted-light observation • set the reflector turret position to none and click the on button for transmitted light. • pull out the push...

  • Page 68

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 62 000000-2071-464 10/2014 v_01 fig. 61 lsm 880 with axio imager.Z2 • via the objective button, select the required objective as follows: − open the graphical pop-up menu by clicking on the objective button. − click...

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    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 63 (2) microscope settings on axio imager for reflected-light observation (epi-fluorescence) • turn on the hbo 100 w or x-cite power supply with switch (fig. 61/ 8 ) • click on the rl re...

  • Page 70

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 64 000000-2071-464 10/2014 v_01 5.1.4 incubation tool to have access to incubator functions to define the atmosphere and temperature, you have to expand the incubation tool by pressing the arrow in the incubation to...

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    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 65 5.2 acquisition tab acquisition contains all functions of the microscope that are needed to set up and control wide-field imaging by bright field or fluorescence, designated in this m...

  • Page 72

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 66 000000-2071-464 10/2014 v_01 5.2.1 configuration functions you can load a predefined acquisition configuration from the experiment manager field (fig. 65) by pressing the load button ( ). All saved acquisition pa...

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    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 67 select the acquisition configuration from the parameters list by highlighting it or search for it by typing its name in the search field. An info menu pops up (fig. 68). Press yes to ...

  • Page 74

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 68 000000-2071-464 10/2014 v_01 5.2.2 smart setup smart setup (fig. 70) provides an intuitive, user-friendly interface for automatically configuring the lsm’s light path for detecting fluorescent dyes. Similar to ca...

  • Page 75

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 69 configure your experiment the fluorescent labels used for staining the sample need to be added to the table in configure your experiment . Dyes are added to this list from a searchabl...

  • Page 76

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 70 000000-2071-464 10/2014 v_01 motifs like in photography, common imaging conditions with typical requirements also exist in laser scanning microscopy. The motifs in smart setup represent presets of scanning parame...

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    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 71 proposals the imaging parameters of the imaging setup tool are defined for the dyes indicated in the panel: configure your experiment. Based upon this set of dyes used for staining th...

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    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 72 000000-2071-464 10/2014 v_01 smart setup displays its proposals side-by-side for optimal comparison. The proposal selected for imaging is highlighted. Key aspects of each proposal are displayed as a bar chart rep...

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    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 73 5.2.3 action buttons action buttons provide control for how the image is acquired (fig. 76). Fig. 76 action buttons they have the following functions, regardless of the imaging mode: ...

  • Page 80

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 74 000000-2071-464 10/2014 v_01 continuous produce images from all active channels and tracks, thereby ignoring the settings for multidimensional acquisition, but taking into account the selected imaging mode. The l...

  • Page 81

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 75 5.2.4 multidimensional acquisition settings in the multidimensional acquisition field (fig. 82), the multidimensionality can be defined by checking the corresponding boxes. Fig. 82 mu...

  • Page 82

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 76 000000-2071-464 10/2014 v_01 fig. 84 multidimensional acquisition tool group – all possible tool tabs displayed note that not all functions are available for all imaging modes. Only those functions that are avail...

  • Page 83

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 77 5.2.5 setup manager – laser open (and close) the laser tool (fig. 86) (only available when the show all tools check box is ticked) by clicking the laser tab in the setup manager tool ...

  • Page 84

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 78 000000-2071-464 10/2014 v_01 diode lasers the lsm 880 can be equipped with diode 440 and / or diode 405 lasers that can be used in continuous wavelength (cw) mode or in pulsed (ps) mode (fig. 88). Selection of th...

  • Page 85

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 79 b. In case the laser is set to on via the power switch on the laser power supply and the key switch on the laser is set to 0, the status report in the laser properties panel shows the...

  • Page 86

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 80 000000-2071-464 10/2014 v_01 maximal in the drop down list next to the power status. The maximal available laser power changes to a higher percentage value and the value is now indicated in red as well as the hig...

  • Page 87

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 81 the active acquisition parameters include all settings of the following tools: − imaging setup − acquisition mode − channels − focus devices and strategy − z-stack − time series − til...

  • Page 88

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 82 000000-2071-464 10/2014 v_01 loops and repetitions are set (in show all mode) in the loops and repetition panel. Use the arrow to open the panel (fig. 94). The number in the loops column states how often a sequen...

  • Page 89

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 83 − autofocus strategies use the center position of a z-stack as the reference position. For a given z-stack go to the center position and if necessary, click center within the center t...

  • Page 90

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 84 000000-2071-464 10/2014 v_01 5.2.7 setup manager – imaging setup the imaging setup tool (only available when the show all tools check-box is ticked) allows new / existing beam path configurations to be created / ...

  • Page 91

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 85 when in channel mode the image acquisition can either be done in one track (all lasers and detectors active simultaneously) or in up to 4 tracks (fig. 97). Configurations for each tra...

  • Page 92

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 86 000000-2071-464 10/2014 v_01 5.2.7.2 filter cube selection the filter cube can be selected from the reflector turret via a graphical pop-up menu when the filter cube button is pressed (fig. 100). There are six po...

  • Page 93

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 87 5.2.7.4 stage control if the stage button is pressed, the stage control panel will appear (fig. 102). By pressing the stop button you can interrupt any stage movement. The move to zer...

  • Page 94

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 88 000000-2071-464 10/2014 v_01 5.2.7.5 focus control if the focus button is pressed, the focus control panel will appear (fig. 103). The z-position display box shows the current z-position. The number can be edited...

  • Page 95

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 89 5.2.7.7 x-cite control when the x-cite on/off button (only available for wf imaging mode) for reflected light of the x-cite or hxp lamp is pressed, the control panel for the light set...

  • Page 96

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 90 000000-2071-464 10/2014 v_01 5.2.7.9 ratio imaging in the show all mode ratio imaging becomes available (only for wf and lsm imaging modes). A click on the arrow next to ratio provides access to the ratio channel...

  • Page 97

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 91 5.2.7.11 control elements of the imaging setup tool this tool displays the hardware settings of the scan head ( lsm tab), the beam path to image with the camera ( wf tab) or the non d...

  • Page 98

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 92 000000-2071-464 10/2014 v_01 a beam splitter is chosen according to the selected laser line(s). It separates the excitation light from the emission signal. These filters reflect the wavelength indicated and trans...

  • Page 99

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 93 activation/deactivation of channels • to activate a channel for imaging, check the check box next to channel icon. To deactivate the channel uncheck this box. • use channel t-ptm (tra...

  • Page 100

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 94 000000-2071-464 10/2014 v_01 • use the slider to set the utilizable laser intensity (recommendation: start at 1 %). − the transmittance of the acousto-optical tunable filter (aotf) changes accordingly. This allow...

  • Page 101

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 95 ratio imaging configuration in the show all mode of the imaging setup tool two additional ratio imaging channels r1 and r2 can be defined. These channels are displayed in addition to ...

  • Page 102

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 96 000000-2071-464 10/2014 v_01 imaging in lambda mode (to be selected in the imaging setup tool). The lambda mode is recording the overall emission from the sample onto a wavelength-dispersive element and is imaged...

  • Page 103

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 97 quasar detector settings use the two sliders to define the spectral detection range of the quasar detector. Resolution : step size can be 3.0, 4.4, 8.9, 17.8, 26.7 or 35.6 nm. Online ...

  • Page 104

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 98 000000-2071-464 10/2014 v_01 wf mode (widefield camera mode) the wf mode (widefield camera mode) displays the current hardware settings for an imaging track using the camera as detection channel. The display chan...

  • Page 105

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 99 5.2.7.12 setup imaging with airyscan detector acquisition, imaging setup, detector activation • to activate the airyscan detector, tick the cha checkbox in the beam path (fig. 118). •...

  • Page 106

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 100 000000-2071-464 10/2014 v_01 multitracking a multitracking acquisition is set up by using the + button (fig. 120). In each track individual settings can be configured for the airyscan or the internal detectors. ...

  • Page 107

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 101 5.2.8 acquisition parameter – acquisition mode in the acquisition mode tool window, parameters for the acquisition can be set (fig. 122). The user interface is adapted depending on t...

  • Page 108

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 102 000000-2071-464 10/2014 v_01 5.2.8.2 scan mode the mode of image acquisition (frame wise, line wise, spot wise) can be selected in the scan mode drop down menu. For the lsm imaging mode you can select between fr...

  • Page 109

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 103 5.2.8.4 averaging and color depth selection you can select the number of image frames you want to average from the averaging number drop down menu (fig. 127). You can select a 2 n x ...

  • Page 110

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 104 000000-2071-464 10/2014 v_01 5.2.8.5 hdr(high dynamic range)-imaging high dynamic range (hdr) creates an image with a depth of 32-bit therefore covering a far broader dynamic range than conventional images (8 to...

  • Page 111

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 105 in the illumination method number of frames is automatically set to 3 (fig. 132). The illumination method changes the laser power (fig. 133) to acquire a series of varying exposures....

  • Page 112

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 106 000000-2071-464 10/2014 v_01 5.2.8.7 acquisition mode functions for lsm the parameters for image acquisition are set using the tool acquisition mode . The following scan modes, which can be selected from the dro...

  • Page 113

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 107 frame mode for lsm when the scan mode frame is active, a frame of variable size is scanned pixel by pixel and line by line. The laser beam is moved over the specimen line by line. Th...

  • Page 114

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 108 000000-2071-464 10/2014 v_01 • select the scan speed from the 19 preset steps via slider or input box. Recommended: 8 for the first scan. A click on the max button sets the maximum speed for the current zoom. − ...

  • Page 115

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 109 in the sum method, the intensity values of all images are added up, without a mean value being calculated. To create the image information using the line average mode, each line (dep...

  • Page 116

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 110 000000-2071-464 10/2014 v_01 frame mode for camera when images are acquired with the camera only frame mode is available. The following additional parameters and be set: objective, frame size, format, bit depth,...

  • Page 117

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 111 line mode in line mode, fluorescent or reflected light along a straight or freely definable line is displayed in the form of an intensity profile. The line is scanned pixel by pixel....

  • Page 118

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 112 000000-2071-464 10/2014 v_01 if the defined free shape curve becomes too complicated or the selected scan speed is too high, the following message appears in the status bar: maximum scanner acceleration exceeded...

  • Page 119

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 113 5.2.8.8 acquisition mode settings for airyscan objective, scan area, zoom the required acquisition settings for the airyscan are set in the acquisition mode tool (fig. 140). • make s...

  • Page 120

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 114 000000-2071-464 10/2014 v_01 airyscan multitrack ilex for best synchronicity in a multitracking acquisition with the airyscan, a so called ilex (interleaved laser excitation) mode is available. This allows the t...

  • Page 121

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 115 5.2.9 acquisition parameter – channels in the channels tool window the following options are available: − switching, activating, deactivating and naming/saving of tracks − defining d...

  • Page 122

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 116 000000-2071-464 10/2014 v_01 5.2.9.1 defining channels and tracks the channels tool lists all tracks in the track selection display area that have been defined in the imaging setup tool. The tracks name and the ...

  • Page 123

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 117 the color of a track can be set or changed selecting a lut from the look up table (lut) drop down menu (fig. 148). The scroll bar allows access to all predefined colors. The color is...

  • Page 124

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 118 000000-2071-464 10/2014 v_01 all saved track configurations will be listed in the load track configuration panel (fig. 151). Select the appropriate one by highlighting it in the recent section, which lists the r...

  • Page 125

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 119 the info window pops up (fig. 153). Press yes to delete the configuration, no to keep the configuration. To save a configuration press the save button (symbolized by the disc icon). ...

  • Page 126

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 120 000000-2071-464 10/2014 v_01 5.2.9.2 ratio channels the parameters for ratio metric imaging can be set when a ratio channel is selected. • click on the button of a ratio channel (e.G. R1 ). • clicking on the req...

  • Page 127

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 121 5.2.9.3 using gaasp detectors and the temperature controlled side pmt for photon counting imaging in the lsm imaging setup tool activate any gaasp detector like big.2, the 32 channel...

  • Page 128

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 122 000000-2071-464 10/2014 v_01 (2) analog (grey value) display in this display, photons are transformed into grey values, which are displayed in discrete intervals in the histograms. Intervals can be calculated by...

  • Page 129

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 123 (3) counting (raw data) format in this display, photons are displayed according to their number (fig. 162). Fig. 162 counting display in photon counting image. Settings were digital ...

  • Page 130

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 124 000000-2071-464 10/2014 v_01 5.2.9.4 channel settings for airyscan the detection settings of the airyscan can be controlled in the channels tool (fig. 163). The pinhole will be set automatically to a correct ope...

  • Page 131

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 125 optimal pixel number and size depending on the detection mode, the required oversampling value for the pixel number and size changes. This is adjusted automatically by using the opti...

  • Page 132

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 126 000000-2071-464 10/2014 v_01 5.2.10 acquisition parameter – focus if the focus tool tab is expanded the focus control tool becomes available (fig. 168). The z-position display box shows the current z-position. T...

  • Page 133

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 127 5.2.11 acquisition parameter – focus devices and strategy focus devices and strategy aims to maintain focus stability in multi-dimensional acquisition experiments. This tool is typic...

  • Page 134

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 128 000000-2071-464 10/2014 v_01 off mode if off is selected, autofocus is inactive and will not be used. If a multi-track acquisition includes a track that employs a camera for data capture, the autofocus mode is a...

  • Page 135

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 129 find offset searches for a reflective surface (the focus reference plane) starting at the z-level of the imaging plane. Next, it copies the increment between the z-levels of the imag...

  • Page 136

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 130 000000-2071-464 10/2014 v_01 here, the autofocus every n timepoint check box is checked and a value of 3 entered in the associated spin box. A focusing event will be executed before the first acquisition and aft...

  • Page 137

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 131 5.2.12 acquisition parameter – stage the following software description applies to systems which are equipped with a motorized scanning stage. If the stage tool window is expanded th...

  • Page 138

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 132 000000-2071-464 10/2014 v_01 5.2.13 acquisition parameter – shuttle and find shuttle and find allows calibration and imaging of sample positions in laser scanning microscopes (lsm) for reproducible repositioning...

  • Page 139

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 133 mounting of cover glasses or tem grids • place the specimen (sample carrier) in the suitable sample holder and fix it: life science cover glass 22x22: − remove the clamping frame (fi...

  • Page 140

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 134 000000-2071-464 10/2014 v_01 calibration the calibration markers consist of one small and a large l-shape marker. The bigger marker is used for coarse orientation, whereas the smaller marker is used for the cali...

  • Page 141

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 135 threshold: high – low : a low threshold for marker detection is used when the dimensions of the correlative l markers cannot be recognized precisely, e.G. When the sample holder is s...

  • Page 142

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 136 000000-2071-464 10/2014 v_01 • if the automatic movement to next position is activated, the stage will move automatically to the next marker (position 2) after clicking accept . Otherwise, the marker 2 has to be...

  • Page 143

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 137 image acquisition and saving • acquire images of your sample as you are used to. • for saving (fig. 183) the images together with coordinates and defined regions of interest or point...

  • Page 144

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 138 000000-2071-464 10/2014 v_01 5.2.14 acquisition parameter – regions the regions tool tab is only available, if the regions checkbox is ticked in the multidimensional acquisition field. If the tool bar is expande...

  • Page 145

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 139 the following tools are available in the tool selection field: selects an existing region in the image. The outline of the region is marked with small rectangles to show it is active...

  • Page 146

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 140 000000-2071-464 10/2014 v_01 when highlighting a roi in the list, the numerical parameters for center x , center y , width and height in pixels will be shown in region feature field. In addition the roi will be ...

  • Page 147

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 141 in this case the scanner movement will be restricted to the bleach region zooming in onto this region. This may result in a less accurate positioning of the region as the definition ...

  • Page 148

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 142 000000-2071-464 10/2014 v_01 5.2.15.1 performing a z-stack using first/last mode press the first/last mode button to open the first/last definition panel. Press the continuous action button and focus onto the sp...

  • Page 149

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 143 the position ( µ m) input box indicates the current position of the focus, no matter if the focus drive or a z-piezo stage insert was used for positioning. The focus is changed in sm...

  • Page 150

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 144 000000-2071-464 10/2014 v_01 5.2.15.2 performing a z-stack using center mode press the center mode button to open the center definition panel (fig. 194). This mode allows setting the range of the z-stack in rela...

  • Page 151

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 145 pressing the center button will define the current focus position (as defined in the position ( µ m) input box) as the center position of the stack. After pressing center the whole r...

  • Page 152

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 146 000000-2071-464 10/2014 v_01 5.2.15.3 performing a z-stack using a z-piezo a z-piezo allows z-stacks to be acquired considerably quicker compared to the focus drive of the microscope stand. The speed advantage d...

  • Page 153

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 147 5.2.15.4 optimize sectioning and step expand the optimize sectioning and step field by pressing the triangle (fig. 196). The optimize sectioning and step function helps to set the op...

  • Page 154

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 148 000000-2071-464 10/2014 v_01 5.2.15.5 correction expand the correction field by pressing the triangle (fig. 198). The refractive index correction input box allows you to consider the different refractive indices...

  • Page 155

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 149 5.2.15.6 z-stack settings for airyscan in case a 3-dimensional acquisition is required, a z-stack (fig. 200) can be defined the same way as when using the internal channels. However,...

  • Page 156

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 150 000000-2071-464 10/2014 v_01 5.2.16 multidimensional acquisition – bleaching when the bleaching tool tab is expanded the bleaching tool becomes available (fig. 202). The bleaching tool is only available in lsm i...

  • Page 157

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 151 if a region is used to define a position for bleaching within the sample, the bleach process can be accelerated by checking the box next to zoom bleach (fast, less accurate) . In thi...

  • Page 158

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 152 000000-2071-464 10/2014 v_01 5.2.17 multidimensional acquisition – tile scan when the tile scan tool tab is expanded the tile scan tool becomes available (fig. 204). This function permits a frame to be created a...

  • Page 159

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 153 tick the checkbox to activate online stitching. After ticking the checkbox the threshold input box becomes available. Enter the desired threshold in % . By pressing add you can mark ...

  • Page 160

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 154 000000-2071-464 10/2014 v_01 marked positions are listed up. This list can be saved and loaded. 5.2.18 scan overview image for tiling pressing the scan overview image… button opens the scan overview image intera...

  • Page 161

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 155 5.2.19 multidimensional acquisition – time series when the time series tool tab is expanded the time series tool becomes available (fig. 211). In the time series tool the parameters ...

  • Page 162

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 156 000000-2071-464 10/2014 v_01 the trigger can be selected from the trigger in pull down menu. Up to 4 triggers are available. The trigger has to be assigned in the firmware of the system. If no trigger is set the...

  • Page 163

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 157 select the timer configuration from the configurations list by highlighting it or search for it by typing its name in the search field. The info window pops up (fig. 216). Press yes ...

  • Page 164

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 158 000000-2071-464 10/2014 v_01 all saved marker configurations will be listed in the load configuration… panel (fig. 220). Select the appropriate one by highlighting it in the recent section, which lists the recen...

  • Page 165

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 159 the info window pops up (fig. 222). Press yes to delete the configuration, no to keep the configuration. To save a marker configuration press the save button (symbolized by the disc ...

  • Page 166

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 160 000000-2071-464 10/2014 v_01 if trigger is selected the trigger in drop down menu becomes available (fig. 226) from which up to 4 triggers can be selected. After pressing start experiment button the system will ...

  • Page 167

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 161 5.2.19.4 end expand the end field by clicking the triangle (fig. 230). In the mode drop down menu (fig. 231) you can select the way the acquisition is triggered. You have the choice ...

  • Page 168

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 162 000000-2071-464 10/2014 v_01 5.2.20 multidimensional acquisition – positions when the positions tool tab is expanded the positions tool becomes available (fig. 234). This function comprises two main applications...

  • Page 169

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 163 5.2.20.2 sample carrier press the sample carrier button to obtain the sample carrier control panel (fig. 236). The last used sample carrier will be displayed in the sample carrier di...

  • Page 170

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 164 000000-2071-464 10/2014 v_01 all saved marker configurations will be listed in the load configuration… panel (fig. 239). Select the appropriate one by highlighting it in the recent section, which lists the recen...

  • Page 171

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 165 select the carrier configuration from the configurations list by highlighting it or search for it by typing its name in the search field. The info window pops up fig. 241). Press yes...

  • Page 172

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 166 000000-2071-464 10/2014 v_01 5.2.20.3 setting individual offset values for positions in position list mode the focus devices and strategy tool contains the controls for maintaining focus stability when using the...

  • Page 173

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 167 5.2.20.5 scan overview image pressing the scan overview image… button opens the scan overview image interactive window (fig. 245). Here you can define the number of tiles by using th...

  • Page 174

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 168 000000-2071-464 10/2014 v_01 5.2.22 multidimensional acquisition – auto save if the auto save tool tab is expanded the auto save tool becomes available (fig. 247). Images that are acquired by clicking the start ...

  • Page 175

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 169 5.2.23 image optimization this section describes an example how to acquire an image, using an excitation wavelength of 561 nm and a fluorescence emission range above 570 nm. Use the ...

  • Page 176

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 170 000000-2071-464 10/2014 v_01 • activate channel 1 via the check box. You may want to assign a different color to the channel which will be taken over for the initial display of the image. Click the color triangl...

  • Page 177

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 171 5.2.23.2 defining the scan parameters • open the acquisition mode tool (fig. 252). • select frame mode and a frame size of 512 x 512 to start with. • the optimal button automatically...

  • Page 178

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 172 000000-2071-464 10/2014 v_01 5.2.23.4 how to optimize image quality as a rule, the first scanned image is not ideal, since the settings of the laser and therefore also the photomultiplier might not match the lig...

  • Page 179

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 173 a similar procedure applies for image optimization if more than one channel is used and simultaneous image acquisition is performed. Make sure the suitable lasers are switched on and...

  • Page 180

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 174 000000-2071-464 10/2014 v_01 raw data sum image, range indicator (fig. 257) in order to fine tune the detection, the raw sum image (source image, left) of the airyscan should be observed carefully. Use the check...

  • Page 181

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 175 5.3 fcs tab the major functions can be selected from the fcs tool group (fig. 258) in the fcs tab. To open saved files click the file button in the menu bar. The fcs tool group consi...

  • Page 182

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 176 000000-2071-464 10/2014 v_01 start experiment action button triggers measurements according to the settings at all defined positions. Stop action button will end an active measurement. Data will be available to ...

  • Page 183

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 177 5.3.1 setup – laser open the laser tool by clicking the laser tab in the setup manager tool group (fig. 259). Close the tool box by clicking again. Select on from the respective pull...

  • Page 184

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 178 000000-2071-464 10/2014 v_01 (1) saving and deleting beam paths you can save a beam path by typing in a memorable name intothe drop down menu selection box and pressing the save button (floppy disc icon). If you...

  • Page 185

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 179 5.3.2.1 light path for 2 channel module (big.2 on dc out port) big.2 has two gaasp detectors that can be used simultaneously to acquire two autocorrelation functions (ac) per each ch...

  • Page 186

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 180 000000-2071-464 10/2014 v_01 the icons are inactive, as the efs are part of the filter cube together with the sbs filter, which has to be replaced manually. The filter are indicated to sw within the filters tool...

  • Page 187

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 181 5.3.2.2 ndd light path (big.2 as ndd) big.2 can be attached to the transmitted (ndd-t) and/or reflected (ndd-r) position (not more than two big.2 modules in can be used on one system...

  • Page 188

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 182 000000-2071-464 10/2014 v_01 the icons are not displayed, because the efs are part of the filter cube together with the sbs filter, which has to be replaced manually. The filter are indicated to sw within the fi...

  • Page 189

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 183 5.3.2.3 light path for 32 channel module the 32 module consists of an array of 32 gaasp detectors plus two side pmts. The side pmt of the longer wavelength region (pmt2) can be used ...

  • Page 190

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 184 000000-2071-464 10/2014 v_01 5.3.2.4 selection of laser lines • press the laser button to display the laser control expansion submenu (fig. 265). Alternatively you have access to the laser lines in the acquisiti...

  • Page 191

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 185 the lasers functionalities for laser control are also available in the acquisition tab (fig. 268). Only that all visible and invisible lasers are displayed together (als applies if s...

  • Page 192

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 186 000000-2071-464 10/2014 v_01 5.3.2.5 pinhole adjustment the adjust pinhole panel allows you to set the pinhole diameter and to automatically align the pinhole position in x and y. • click on the adjust pinhole t...

  • Page 193

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 187 the pinhole is adjusted using a dye solution. The general approach is to move the pinhole cyclically in x and y until the intensity maximum is found. For each excitation wavelength a...

  • Page 194

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 188 000000-2071-464 10/2014 v_01 • you can also alter the position by typing into the input boxes or using the sliders or scroll arrows. If both coordinates are changed, the system will regard this as a new alignmen...

  • Page 195

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 189 procedure to adjust pinholes • mount a carrier with a suitable dye solution and orient the sample stage. Select the test sample. • select the beam path or the method with attached be...

  • Page 196

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 190 000000-2071-464 10/2014 v_01 5.3.3 setup – acquisition measure time [s] the measure time (fig. 271) is the period of one measurement. As a rule of thumb the measurement time should be about 1000 x the diffusion ...

  • Page 197

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 191 • set the measure time . If you do not have any idea about the behavior of your sample, start with a measurement time of about 10 seconds. Depending on the signal-to-noise ratio of y...

  • Page 198

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 192 000000-2071-464 10/2014 v_01 5.3.4 setup – fit the fit tool allows you to load a model to be used for the measurement as well as to define new models. Loading a predefined method the fit tool menu allows you to ...

  • Page 199

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 193 − upper limit : defines the upper limit value tolerated from a fit; if this value is exceeded, the fit is rejected and another (global) minimum is searched for. The default value dep...

  • Page 200

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 194 000000-2071-464 10/2014 v_01 5.3.4.1 defining a new model if the define button is pressed, the define model panel will open. It is used to define model equations to which measured data can be fitted. • click on ...

  • Page 201

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 195 5.3.4.2 correlation options in correlation you can assemble a model with predefined terms. • press the correlation tab to activate the options for assembling a correlation model (fig...

  • Page 202

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 196 000000-2071-464 10/2014 v_01 5.3.4.3 model equations in the following, the mathematical equations of the correlation functions and the fit equations will be more detailed. The acquired correlation functions must...

  • Page 203

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 197 definition of the cross-correlation function the formalism for the cross-correlation function is identical to the auto-correlation function, with the exception that the signal in one...

  • Page 204

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 198 000000-2071-464 10/2014 v_01 a. Amplitudes the amplitude of the correlation function is influenced by the offset, background and the number of particles in dependence of the geometric factor. The amplitude is al...

  • Page 205

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 199 a.4 amplitude a 1 ) 0 ( − = = g n a γ (5a) where γ is the geometric factor accounting for the point spread function (psf) and n the mean number of particles. In the fcs software γ ca...

  • Page 206

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 200 000000-2071-464 10/2014 v_01 the following relations exist: d r d ⋅ = 4 2 ω t for 1 photon excitation (5e) d r d ⋅ = 8 2 ω t for 2 photon excitation (5f) r z s ω ω = (5g) equations 5e or 5f, dependent on the exc...

  • Page 207

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 201 b. Anti-bunching terms anti-bunching is the phenomenon that a molecule cannot produce emitted photons as long as it stays in the excited state. Hence during the transition time requi...

  • Page 208

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 202 000000-2071-464 10/2014 v_01 k 1 and t k1 are fit parameters; k 1 is a fixed parameter and must be user defined; κ 1 is either a fit parameter or can be fixed. Note, fixing k 1 and κ 1 to "1" result s in a simpl...

  • Page 209

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 203 c. Bunching terms bunching is the phenomenon of a burst of photons during a certain time interval, the duration of which is determined by photo-physical processes including triplet, ...

  • Page 210

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 204 000000-2071-464 10/2014 v_01 blinking is based on the phenomenon that the electron distribution over conjugated systems can change in dependence on the local environment, for example changes in the ph, which wil...

  • Page 211

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 205 c.4 independent triplet and blinking in this case the terms are just representatives for two dependent bunching terms that are linked by multiplication. Note that the triplet fractio...

  • Page 212

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 206 000000-2071-464 10/2014 v_01 c.6 double stretched exponential - bunching this is a double exponential function, where the exponentials are added. 2 2 2 1 1 1 ) ( 2 2 ) ( 1 1 1 ) ( κ κ t t t t k k k k k e k k e k...

  • Page 213

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 207 d. Diffusion terms diffusion is driven by brownian motion. We can distinguish translational, rotational and flow diffusion. D.1 rotational diffusion in the most general form, rotatio...

  • Page 214

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 208 000000-2071-464 10/2014 v_01 a r r a r r e c e c r g r r − ⋅ + ⋅ ⋅ + = − − 1 ) ( 1 ) ( 2 , 2 1 , 1 2 1 t t t t t normalized (8f) with r a being the amplitude, c 1 and c 2 relative amplitudes, r 1 and r 2 frequen...

  • Page 215

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 209 d.2 translational diffusion in its general form, translational diffusion is defined as: ⋅ + ⋅ + Φ = ∑ = 3 1 2 1 2 , , 2 1 1 1 1 ) ( i e i d e i d i d d i d i s g α α t t t t t with t...

  • Page 216

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 210 000000-2071-464 10/2014 v_01 t d,i are fitted parameters. They can be converted to diffusion coefficients d i using formulas 5e or 5f. The fcs software allows you to directly fit to d i values, but in this case ...

  • Page 217

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 211 note that the brightness contributes as the square to the correlation function, in other words a double as bright molecule will contribute 4 fold more. Therefore, the fitted number o...

  • Page 218

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 212 000000-2071-464 10/2014 v_01 d.3 flow flow signifies active transport either via cytoplasmic movement or directed transport. If flow occurs in the absence of translational diffusion, the term is defined as follo...

  • Page 219

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 213 5.3.4.4 definition of a model • to define a model press the term tab of the term you want to be part of your final correlation equation. The tab will be highlighted in blue. Press th...

  • Page 220

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 214 000000-2071-464 10/2014 v_01 background • you can set an offset by setting a value in the background selection box (fig. 277). Either type in a value or use the arrows. The description box gives you information ...

  • Page 221

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 215 antibunching you can select the anti-bunching term in its normalized form by checking the normalized check box or in its non-normalized form by unchecking the box (fig. 279). You can...

  • Page 222

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 216 000000-2071-464 10/2014 v_01 triplet the triplet represents bunching terms that are exponential decay functions. You can select the triplet term in their normalized form by checking the normalized check box or i...

  • Page 223

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 217 rotation you can choose between normalized and non- normalized rotation functions by selecting / dese- lecting the normalized check box (fig. 283). You can select the dependent antib...

  • Page 224

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 218 000000-2071-464 10/2014 v_01 you can choose between symmetric rotation (1 exponential term) and asymmetric rotation (double exponential term) by selecting / deselecting the asymmetric check box (fig. 285). If th...

  • Page 225

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 219 translation you have the following possibilities for setting parameters (fig. 286): • you can either fit to fractions (used normally when no brightness differences are observed betwe...

  • Page 226

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 220 000000-2071-464 10/2014 v_01 flow you can determine, if you want to fit to the diffusion time or to the velocity directly by un- ticking or ticking the velocity check box (fig. 287). In the latter case you have ...

  • Page 227

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 221 stretched exponential you can select between bunching and anti- bunching terms by pressing the bunching or antibunching buttons, respectively (fig. 288). You can select between 1 (mo...

  • Page 228

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 222 000000-2071-464 10/2014 v_01 5.3.4.5 pch in the pch (photon counting histogram) panel you can determine concentrations and the molecular brightness of molecules. • click on the pch tab to open the pch control pa...

  • Page 229

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 223 5.3.4.6 formula the formula panel allows you to define your own fit equation. • press the formula tab to open the formula panel (fig. 290). Click any other tab to leave the panel. In...

  • Page 230

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 224 000000-2071-464 10/2014 v_01 5.3.5 setup – correlator in the correlator tool you can define the fit and acquisition parameters for the measurement. • click on the correlation tab to open the correlation panel (f...

  • Page 231

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 225 together and normalized. The normalization result is defined as: (result x received clocks)/(pulse 2 ) for auto-correlation or (result x received clocks)/(pulsea x pulseb) for cross-...

  • Page 232

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 226 000000-2071-464 10/2014 v_01 (3) pch in pch panel the binning time used for the photo counting histogram can be selected. • click on the pch tab to open the pch panel (fig. 293). You can either check the automat...

  • Page 233

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 227 please note that the cut off count rate is defined as a value exceeding the average count rate during a certain measurement period (bin window) by a certain percentage. Thus, the con...

  • Page 234

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 228 000000-2071-464 10/2014 v_01 5.3.6 navigation – stage • click on the stage tool tab to open the stage panel (fig. 296). The compass rose allows you to move the scanning stage in the direction of the arrows that ...

  • Page 235

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 229 5.3.7 navigation – sample carrier the sample carrier tool allows the definition and orientation of a sample carrier, so that single wells can be approached automatically. • click on ...

  • Page 236

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 230 000000-2071-464 10/2014 v_01 in order to delete a configuration, press the delete button ( cross symbol). This will open the delete configuration… window (fig. 302). Select the configuration you want to delete b...

  • Page 237

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 231 5.3.8 navigation – focus the function of the focus tool allows you to position the focus in z direction. • press the focus tool tab to open the focus tool (fig. 304). Press anywhere ...

  • Page 238

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 232 000000-2071-464 10/2014 v_01 proceed as follows: • make sure that the front lens of the water immersion objective is wetted by a drop of water. Use fluorescence-free double-distilled water, since otherwise the i...

  • Page 239

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 233 • set up scan mode in the lsm acquisition mode tool (fig. 306). Choose line scan, highest scan speed, 512 pixels and zoom 1 for convenience. • set up scan control in the lsm channels...

  • Page 240

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 234 000000-2071-464 10/2014 v_01 • press continuous to start a continuous scan. Press the line scan tab (fig. 308). • the diagram (fig. 309) shows a line with a bump, which is the result of reflected light on the op...

  • Page 241

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 235 • continue moving upwards slowly. The line position will reach its maximum (fig. 311). • having slowly moved over a short distance the line position will decline again to a minimum a...

  • Page 242

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 236 000000-2071-464 10/2014 v_01 5.3.9 multidimensional acquisition tools – time series the time series tool is only available if the time series box in the tab activation area is checked (fig. 338). In the time ser...

  • Page 243

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 237 if the kinetic will last longer than one day, a warning is displayed (fig. 317). Click ok to continue. Define the following variables: time delay between consecutive data points = pi...

  • Page 244

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 238 000000-2071-464 10/2014 v_01 5.3.10 multidimensional acquisition tools – positions in the positions tool you can select the carrier and the sample or laser position by the stage or by the scanners. • click on th...

  • Page 245

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 239 if the xy stage button is activated, all positioning will be done via the scanning stage. This mode corresponds to the sample carrier mode, if single position is selected. Regardless...

  • Page 246

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 240 000000-2071-464 10/2014 v_01 (2) sample carrier the sample carrier is used to approach a single well or multiple wells sequentially in an automatic mode. • click on the sample carrier tab to open the sample carr...

  • Page 247

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 241 • click on the corresponding chamber in the sample carrier window (fig. 321). − this chamber will be flashing up briefly. The stage is now oriented. Note that there is no further ind...

  • Page 248

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 242 000000-2071-464 10/2014 v_01 single position only a single well can be activated. The scanning stage will automatically move to the activated well without the need to trigger a measurement. To select the chamber...

  • Page 249

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 243 multiple positions • select from the working direction drop down menu an entry other than single position . • select as many individual well positions as required (all are selectable...

  • Page 250

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 244 000000-2071-464 10/2014 v_01 • move the cursor to the desired position in the scan image (drag and drop). Press the add button to add the position marked with the crossline to the positions list (fig. 324). A cr...

  • Page 251

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 245 the x-, y- and z-coordinates of the selected positions are only stored in the positions table and not in the scanned image (stack). Accordingly, all the marked positions in all z-pla...

  • Page 252

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 246 000000-2071-464 10/2014 v_01 to compensate for scan zoom and scan speed go to the acquisition mode too of the lsm online acquisition tool group. Use a sample with high contrast, like "convolaria", and focus. Act...

  • Page 253

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 247 5.3.11 navigation – incubation open the incubator tool by clicking the incubator tab in the navigation tool group (fig. 331). You will have access dependent on the attached equipment...

  • Page 254

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 248 000000-2071-464 10/2014 v_01 5.3.12 multidimensional acquisition tools – information on experiment • click on the information on experiment tab to display a detailed description of the highlighted method includi...

  • Page 255

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 249 5.3.13 action buttons for fcs the action buttons are used to trigger measurements and to adjust the measurement conditions. The following buttons are available (fig. 333): − new: ope...

  • Page 256

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 250 000000-2071-464 10/2014 v_01 5.3.14 method tool the method tool allows to store ( floppy disc button), load ( open file button) and to delete ( cross button) methods. (1) storing a method the store button ( flop...

  • Page 257

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 251 (2) loading a method the load button ( open file symbol) will allow you to load a pre-defined method. The configurations will be displayed; however, hardware settings change only if ...

  • Page 258

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 252 000000-2071-464 10/2014 v_01 (3) deleting a method the delete button ( cross symbol) allows you to delete a method from the database. • click on the delete button. A delete configuration… window will appear (fig...

  • Page 259

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 253 5.3.15 activation tab area in the activation area you can select certain tabs to be displayed in the tool area or no by just checking the corresponding boxes (fig. 338). Those tools ...

  • Page 260

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 254 000000-2071-464 10/2014 v_01 5.3.17 count rate • click the count rate button to open the count rate window (fig. 339). A) display select one of the display modes count rate , correlation or cpm (counts / molecul...

  • Page 261

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 255 • click the cpm button to open the counts per molecule per second (cpm) (fig. 342). This value is constantly updated and calculated each 0.5 ms if averaging is switched off . The blu...

  • Page 262

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 256 000000-2071-464 10/2014 v_01 5.3.18 x-y-z scan the x-y-z scan panel allows you to perform scans in the x, y and z directions and combinations thereof. Especially in the z direction, it helps to position the focu...

  • Page 263

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 257 please note, that x and y scans are performed by the stage regardless if the scanner button in the measurement tool group is pressed or not. Z scans are performed by the nosepiece. S...

  • Page 264

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 258 000000-2071-464 10/2014 v_01 the data of the count rate scan window can either be copied to the clipboard or written to a text file. A click in the count rate scan window with the right mouse button will open th...

  • Page 265

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 259 (1) manual focusing on the membrane • acquire an image of the cell. • place the focus above the cell and scan continuously. • focus slowly down until you see a fluorescence signal. •...

  • Page 266

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 260 000000-2071-464 10/2014 v_01 5.3.21 taking fcs measurements • click on the new document button to open a new fcs diagram in an image container . New diagrams per session will be numbered starting with 0 (fig. 34...

  • Page 267

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 261 the fcs diagram contains five layers, which can be selected by pressing the corresponding tab: − correlation: displays the count race trace, correlation function, photon counting his...

  • Page 268

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 262 000000-2071-464 10/2014 v_01 pressing new will open an empty fcs diagram. To generate data in the fcs diagram you have to trigger a measurement by pressing the snap or start experiment button. After the measurem...

  • Page 269

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 263 5.3.21.1 start and end of a measurement fig. 351 fcs correlation window during data acquisition after setting the parameters the measurement can be run. • click on the snap or start ...

  • Page 270

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 264 000000-2071-464 10/2014 v_01 (1) positioning in solution • align a carrier in the carrier position tool. Focus into the solution. • go to the measure tool and the acquisition panel. Choose current position or sa...

  • Page 271

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 265 the microscope control tool contains the following functions: hal on/off button for transmitted light: if the button is pressed, the control panel appears and the shutter automatical...

  • Page 272

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 266 000000-2071-464 10/2014 v_01 home and load positions can also be accessed from the z-position slider by pressing the h or l buttons. The z-position can be altered by grapping and sliding the plane or by using th...

  • Page 273

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 267 5.3.21.2 loading stored data files to load stored data files you can use the file menu from the main toolbar (fig. 356). • go to file and than choose open . This will open the open d...

  • Page 274

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 268 000000-2071-464 10/2014 v_01 the image fcs diagram consists of four data layers: − fcs correlation − fcs coincidence − fcs fit − fcs print each layer will display the data as they were stored last. As default, t...

  • Page 275

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 269 fig. 359 image container with fcs diagram containing loaded data and fcs correlation selected the result table below the diagrams displays the measuring results. The width of the col...

  • Page 276

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 270 000000-2071-464 10/2014 v_01 selection of lines and properties of the table can be defined by pressing the right mouse button, when the cursor is within the table. This will open the result table context menu (f...

  • Page 277

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 271 color assignment to fcs curves • select those fcs curves in the result table with the right mouse button and pressing the ctrl key, for which you want to assign a new color. • click ...

  • Page 278

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 272 000000-2071-464 10/2014 v_01 reuse with a click on the reuse button, the configuration used to record the selected measurement will be loaded. Reload with reload the current active measurement will be reopened. ...

  • Page 279

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 273 5.3.21.5 image view controls – table tab the table tab (fig. 365) chosen from the option tool area displays several options: − in the average display window you can select, how the d...

  • Page 280

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 274 000000-2071-464 10/2014 v_01 (1) count rate trace in the count rate diagram the count rate(s) (cr) in khz is plotted vs. Running time. If a cross correlation set-up is used, the count rate trace for each channel...

  • Page 281

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 275 (2) correlation g( t ) the diagram shows the correlation functions for each activated channel. The zoom function can be used to display certain diagram areas of interest in an enlarg...

  • Page 282

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 276 000000-2071-464 10/2014 v_01 fig. 368 the correlation curve window in normal (upper graph) and normalized (lower graph) display.

  • Page 283

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 277 (3) pulse distance histogram this diagram shows the frequency plotted against the times elapsed between two subsequent pulses (or: photons recorded from the detector). To obtain this...

  • Page 284

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 278 000000-2071-464 10/2014 v_01 • choosing abscissa logarithmic or ordinate logarithmic will display the abscissa or ordinate in a logarithmic scaling. • the line width allows you to select the thickness of the gra...

  • Page 285

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 279 (2) export with export , the measured data will be exported in a confocor 1-compatible format and can be handled using the fcs access fit program. In the export window you can enter ...

  • Page 286

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 280 000000-2071-464 10/2014 v_01 5.3.21.7 coincidence the coincidence analysis generates a two-dimensional color-coded count rate histogram (fig. 374) in this diagram. During the measurement the count rates in chann...

  • Page 287

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 281 5.3.21.8 fit • click on the fit tab to activate the fit display mode (fig. 375). In general this menu allows you to work with already measured and probably also fitted correlation cu...

  • Page 288

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 282 000000-2071-464 10/2014 v_01 for fitting to a pch model the upper graph (fig. 376) shows the pch fit diagram curves (frequency against count rate per bin) with the overlaid fit graph. If an experiment is loaded ...

  • Page 289

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 283 • click on the diagram with the right mouse button to open the context menu for the fit correlation (fig. 377) respectively fit pch diagrams (fig. 378). • a click on the reset diagra...

  • Page 290

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 284 000000-2071-464 10/2014 v_01 • select normalize to display the correlation curves in a normalized form (fig. 379). The amplitude of the diffusion contribution will be normalized to 2, not the total amplitude, if...

  • Page 291

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 285 the context menu of the fit deviation diagram (fig. 380) can be used to reset the zoom to its original value, copy the data, and adjust the display. • click on the diagram with the r...

  • Page 292

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 286 000000-2071-464 10/2014 v_01 lower and upper limits are only displayed if selected. To select, click with the right mouse button into the result table to open the context menu (fig. 382). Fig. 382 fit table with...

  • Page 293

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 287 (3) channel display field the channel display field shows the active channel (selected name and color), for which the fit all button applies. It is the last selected channel. If for ...

  • Page 294

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 288 000000-2071-464 10/2014 v_01 the global link status of a parameter is displayed in the link column. By clicking with the left mouse button in the respective field, a button tab becomes available. Use the arrow h...

  • Page 295

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 289 (5) state display box the state display box (fig. 385) above the fit action buttons informs you about the fitting conditions. Whenever some inconsistencies are present, e.G having to...

  • Page 296

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 290 000000-2071-464 10/2014 v_01 5.3.21.9 print click on the print tab to activate the print diagrams display mode (fig. 387). The print diagrams window shows an overview of the results presented in tables and / or ...

  • Page 297

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 291 fig. 387 print panel.

  • Page 298

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 292 000000-2071-464 10/2014 v_01 5.3.21.10 information fig. 388 information panel the information panel (fig. 388) contains three blocks: name , comment and acquisition . Names: this is an entry box, where you can i...

  • Page 299

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 293 5.4 processing tab 5.4.1 general structure of the processing tab click on the processing tab to have access to the processing functions. The list of available processing tool will be...

  • Page 300

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 294 000000-2071-464 10/2014 v_01 all processing tools can be used with stored and reloaded images or with newly acquired images (that need not be stored before processing). Please note that for palm images, although...

  • Page 301

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 295 the processing tool tab (fig. 390) is structured into different fields. The top field or header (just beneath the main tool tab area) contains the apply button. The button is only ac...

  • Page 302

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 296 000000-2071-464 10/2014 v_01 processing functions do not contain additional options and the field remains empty. Only those functions will be displayed that match the dimensions of the image parameters and that ...

  • Page 303

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 297 5.4.2 processing – airyscan processing licensing instrument standard optional lsm elyra lightsheet z.1 tool accepts z-stacks and time series and combinations of both. See full descri...

  • Page 304

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 298 000000-2071-464 10/2014 v_01 manual • for manual processing, deactivate the auto checkbox (fig. 394). • in the preview window you can check again whether the right image type is selected. Note that this preview ...

  • Page 305

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 299 batch • for batch processing press the run batch button (fig. 396) and select the respective directory and files from the file selection window which opens at the top. • after confir...

  • Page 306

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 300 000000-2071-464 10/2014 v_01 5.4.3 processing – maximum intensity projection licensing instrument standard optional lsm elyra lightsheet z.1 tool accepts z-stacks and time series (incl. Combinations with several...

  • Page 307

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 301 5.4.4 processing – color-coded projection licensing instrument standard optional lsm elyra lightsheet z.1 tool accepts z-stacks and time series (incl. Combinations with several other...

  • Page 308

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 302 000000-2071-464 10/2014 v_01 5.4.5 processing – image calculator licensing instrument standard optional lsm elyra lightsheet z.1 tool accepts all types of image data recorded with the listed instruments. See ful...

  • Page 309

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 303 for an overview of the available more advanced operators, please refer to the formula panel that is shown in a pop-up window after clicking the operators… button (fig. 400). By highl...

  • Page 310

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 304 000000-2071-464 10/2014 v_01 5.4.6 processing – average licensing instrument standard optional lsm elyra lightsheet z.1 tool accepts all types of image data recorded with the listed instruments. See full descrip...

  • Page 311

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 305 5.4.7 processing – filter licensing instrument standard optional lsm elyra lightsheet z.1 tool accepts all types of image data recorded with the listed instruments. It is generally a...

  • Page 312

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 306 000000-2071-464 10/2014 v_01 the effect of the filter increases with the matrix size. This matrix / kernel size can be set for each dimension with the three sliders kernel size x , kernel size y , and kernel siz...

  • Page 313

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 307 band filter with the band filter (fig. 407), the original image is filtered with a "band blocker" filter. It filters out a certain frequency band with the position set by center , wi...

  • Page 314

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 308 000000-2071-464 10/2014 v_01 5.4.8 processing – linear unmixing licensing instrument standard optional lsm elyra lightsheet z.1 tool accepts lambda stacks and multi-channel image data (single images and multi- d...

  • Page 315

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 309 • pressing the select button loads the active image from the image display and displays it as input image. Only lambda stacks (only available in lsm imaging modes) or multi-channel i...

  • Page 316

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 310 000000-2071-464 10/2014 v_01 • select the sample-specific fluorescence dye reference spectra from the spectra database (fig. 411). • assign appropriate colors to the fluorescence channels by clicking on the colo...

  • Page 317

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 311 weighted unmixing: when this option is checked, spectral channels with high noise contribute less to the unmixing result. This option includes a statistical analysis of the signal-re...

  • Page 318

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 312 000000-2071-464 10/2014 v_01 multichannel unmixing: when this option is chosen. The unmixing algorithm is applied to a multi-channel image (up to 10 channels) without the use of reference spectra. The calculatio...

  • Page 319

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 313 5.4.9 processing – ion concentration licensing instrument standard optional lsm elyra lightsheet z.1 tool accepts single-channel or multi-channel, multi-dimensional data (typically t...

  • Page 320

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 314 000000-2071-464 10/2014 v_01 image scaling spin boxes to set the min and max in the output image pull down menus for to set three different calibration options, according to the dyes (single wavelength, vs ratio...

  • Page 321

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 315 5.4.9.1 single wavelength dyes – offline calibration • subtract background/ autofluorescence image from raw images to obtain better raw data to start with (fig. 417). • perform equat...

  • Page 322

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 316 000000-2071-464 10/2014 v_01 5.4.9.2 ratiometric dyes • fura-2, indo-, snarf, cameleon, ratiometric pericam, phluorin. • display fluorescence ratio r over time • display fluorescence ratio r corrected for backgr...

  • Page 323

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 317 ratiometric dyes - calibration (fig. 419) • subtract background/autofluorescence images from raw images to obtain r korr [=f 1 -f 1background )/(f 2 -f 2background )] when calibratio...

  • Page 324

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 318 000000-2071-464 10/2014 v_01 ratiometric dyes - equation calibration (grynkiewicz) (fig. 420) fura-2, indo-1, k d (dissociation constant) taken from literature r min : derived from ion-free state of the dye (e.G...

  • Page 325

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 319 5.4.10 processing – correlation licensing instrument standard optional lsm elyra lightsheet z.1 tool accepts potentially all types of image files. See full description of tool for de...

  • Page 326

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 320 000000-2071-464 10/2014 v_01 5.4.11 processing – modify series licensing instrument standard optional lsm elyra lightsheet z.1 tool accepts single image files (for concatenation) and multi-dimensional image seri...

  • Page 327

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 321 5.4.12 processing – hdr-imaging licensing instrument standard optional lsm elyra lightsheet z.1 tool accepts image data recorded with the optional feature " hdr " of the acquisition ...

  • Page 328

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 322 000000-2071-464 10/2014 v_01 the sampling mode is a meant to improve the image display as it equalizes the differences between the very high and the very low intensities in the image. However, it is not suitable...

  • Page 329

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 323 after acquisition, the grey dynamics of the image are automatically linearized (see description above) to match the range of the output display. This step visibly intensifies weak si...

  • Page 330

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 324 000000-2071-464 10/2014 v_01 during data recording with the illumination method, masks are applied to protect the sample from laser irradiation. The respective rois are displayed in an additional image (fig. 428...

  • Page 331

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 325 5.4.13 processing – stitch licensing instrument standard optional lsm elyra lightsheet z.1 tool accepts image data recorded data recorded with the acquisition tool tile scan (incl. C...

  • Page 332

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 326 000000-2071-464 10/2014 v_01 5.4.14 processing – palm licensing instrument standard optional lsm elyra lightsheet z.1 tool exclusively accepts palm data. See full description of tool set for details. The process...

  • Page 333

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 327 5.4.14.1 palm – convert to image the convert to image function allows converting the vector map of centroids to be transformed to a bona fide image. Select the convert to image tab t...

  • Page 334

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 328 000000-2071-464 10/2014 v_01 5.4.14.2 palm – import palm molecules the import palm molecules function allows importing palm molecule tables saved as text files (extension .Txt) and reconstruct a palm image from ...

  • Page 335

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 329 5.4.14.3 palm – localization precision select localization precision to attach a localization precision look up table (lut) to an experimental psf (fig. 437). Highlight the image wit...

  • Page 336

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 330 000000-2071-464 10/2014 v_01 5.4.14.4 palm – palm the palm function allows setting filters for peak finding and choosing a fit model for the localization calculations. Select the palm tab to select the palm proc...

  • Page 337

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 331 − account for overlap (multi-emitter): multi- emitter events will be fitted taking into account the overlap. If selected, the maximum cluster size slider and input box become availab...

  • Page 338

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 332 000000-2071-464 10/2014 v_01 the peak mask size filter defines the radius in pixel units for a detected peak intensity that will be used to cut out the peak and put the peak into a new coordinate system. The siz...

  • Page 339

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 333 if the discard overlapping molecules was selected from the settings drop down menu, all identified peaks with overlapping circles will be indicated in red and discarded (fig. 449). I...

  • Page 340

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 334 000000-2071-464 10/2014 v_01 the peak intensity to noise filter defines the threshold of the signal strength of an identified peak over background, at which the peak is regarded to be genuine. The filter works a...

  • Page 341

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 335 expand the localizer field to have access to the fit model used to localize peaks (fig. 452). Three fit models are available that can be selected by the fit model drop down menu (fig...

  • Page 342

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 336 000000-2071-464 10/2014 v_01 if the average before localization check box remains inactive, every peak (event) will be localized independently and is regarded as being derived from one molecule. Later on, peaks ...

  • Page 343

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 337 expand the grouping field to have access to the grouping tool (fig. 458). You can set two filters to be taken into consideration when peaks (events) are grouped: − off gap. If peaks ...

  • Page 344

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 338 000000-2071-464 10/2014 v_01 5.4.14.5 palm – remove palm outliers the remove palm outliers function allows removing all molecules that within a defined radius will have less than the specified numbers of neighbo...

  • Page 345

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 339 5.4.15 processing – particle tracking licensing instrument standard optional lsm elyra lightsheet z.1 tool was exclusively developed for tracking molecules or particles having a dot-...

  • Page 346

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 340 000000-2071-464 10/2014 v_01 once the settings are complete press apply to obtain the trajectories of the single molecules (fig. 466). Please note, that particle tracking only works for 2d data sets. Fig. 466 im...

  • Page 347

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 341 5.4.16 processing – experimental psf licensing instrument standard optional lsm elyra lightsheet z.1 tool accepts z-stacks. See full description of tool for details. This function al...

  • Page 348

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 342 000000-2071-464 10/2014 v_01 the psf sampling pull down menu allows you to interpolate the result to different degrees. Since the resulting psf is an average of many psfs corresponding to the localized beads and...

  • Page 349

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 343 feature size sets the mask size that is used to cut out the bead and to create the experimental psf . The extensions are displayed in the preview tab of the image container as circle...

  • Page 350

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 344 000000-2071-464 10/2014 v_01 once settings have been completed the preview window displays beads that are not taken into account as red circles and those that are used in white circles (if channel color is grey)...

  • Page 351

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 345 5.4.17 processing – structured illumination licensing instrument standard optional lsm elyra lightsheet z.1 tool accepts data recorded in sim mode on elyra. In data sets containing o...

  • Page 352

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 346 000000-2071-464 10/2014 v_01 5.4.17.1 structured illumination – fourier transform the fourier transform tool computes and displays the fourier transform of the image (frequency space). Note that the fourier imag...

  • Page 353

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 347 5.4.17.2 structured illumination – structured illumination the structured illumination function allows automatic or manual processing of a set of structured images obtained by phase ...

  • Page 354

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 348 000000-2071-464 10/2014 v_01 the run batch button will turn into a stop batch button (fig. 481) once pressed. If the button is pressed, batch processing will be stopped, no matter if apply was pressed or not and...

  • Page 355

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 349 if the stack is large, available memory might be limited. In this case a warning is issued, recommending the usage of the 3d large mode (fig. 486). In this case, the stack is subdivi...

  • Page 356

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 350 000000-2071-464 10/2014 v_01 if the max.Isotrop checkbox is ticked, the software will compute the high resolution image with the same resolution in every direction that is it does not use the highest frequencies...

  • Page 357

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 351 the preview tab shows the input image and a preview of the output image. By pressing default the current parameter values will be stored as default values. By pressing reuse the last...

  • Page 358

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 352 000000-2071-464 10/2014 v_01 5.4.17.3 structured illumination – tirf- filtered this function works for sim images (stack) in combination with a tirf image. The lowest frame (first frame) of the stack, preferenti...

  • Page 359

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 353 the link drop down menu available for each channel displays all channels of the sim input image (fig. 500). You can now link the tirf channel to one of the sim channels by highlighti...

  • Page 360

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 354 000000-2071-464 10/2014 v_01 5.4.18 processing – channel alignment licensing instrument standard optional lsm elyra lightsheet z.1 tool accepts multi-color image data (including multi-dimensional data sets). See...

  • Page 361

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 355 if the fit check box is ticked and the input 2 checkbox inactive, than the software will align all channels of input image having the same format to the first listed channel of the s...

  • Page 362

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 356 000000-2071-464 10/2014 v_01 lateral (fig. 503) is used to correct for linear lateral and axial drifts, that is in all directions. Linear alignments are performed in the x, y and z direction, when the applied bu...

  • Page 363

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 357 using the channel alignment tool for drift corection the following procedure can be used to remove lateral drift in an inage: • duplicate the time series. Select the time series in t...

  • Page 364

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 358 000000-2071-464 10/2014 v_01 • apply drift correction. In the channel alignment processing function select the reference time series as input 1 and the original time series as input 2 . Check the fit box and the...

  • Page 365

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 359 when the save default button is pressed, the present coefficients will be stored and can be recalled by pressing the restore default button, which becomes active once the save defaul...

  • Page 366

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 360 000000-2071-464 10/2014 v_01 in either case, the other loaded image does not play a role. In the coefficient table the channel ( ch ) assignment column is replaced by an identity ( id ) column that numbers the c...

  • Page 367

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 361 5.4.19 processing – lightsheet processing licensing instrument standard optional lsm elyra lightsheet z.1 see full description of tool set for details. The lightsheet processing modu...

  • Page 368

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 362 000000-2071-464 10/2014 v_01 mean fusion : the average intensity for each pixel is calculated using the values from both illumination sides. Maximum fusion : the intensity of each pixel of both illumination imag...

  • Page 369

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 363 after pressing apply , a new .Czi file will be generated. The original file name will be extended with "_dual side fusion". For this resulting dataset, the illumination dimension is ...

  • Page 370

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 364 000000-2071-464 10/2014 v_01 5.4.19.2 multiview processing 5.4.19.2.1 deconvolution for the multiview datasets acquired with the lightsheet z.1, deconvolution is an available option (fig. 518). The following dat...

  • Page 371

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 365 fast iterative: the "fast iterative" method is an iterative restoration method that uses only one iteration per convolution step (see meinel, e.S.: origins of linear and nonlinear re...

  • Page 372

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 366 000000-2071-464 10/2014 v_01 adjust strength checkbox: if you have selected the nearest neighbor algorithm or fast iterative, the set strength manually checkbox is always activated. With the other algorithm opti...

  • Page 373

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 367 to edit any of the input boxes, press the override button. To get back to the settings provided by the software, press the master reset button. In the microscope parameters section a...

  • Page 374

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 368 000000-2071-464 10/2014 v_01 5.4.19.2.2 registration options the registration options check-box next to multiview processing opens the interface to define the settings for the registration parameters , the input...

  • Page 375

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 369 5.4.19.2.2.1 landmark alignment for the landmark alignment (fig. 522), the sample must have been embedded with appropriate fluorescent beads (fiducials) which can be recognized by th...

  • Page 376

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 370 000000-2071-464 10/2014 v_01 for mean fusion and maximum fusion the checkbox fusion subset in x with preview can be activated (fig. 523). With these controls it is possible to define which area of the left and t...

  • Page 377

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 371 ignore sample : this option activates an additional function to find structures within the sample that were incorrectly labeled as beads (fiducials). These structures are not taken i...

  • Page 378

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 372 000000-2071-464 10/2014 v_01 5.4.19.2.2.2 intensity based alignment the intensity based alignment option does not need any fluorescent beads (fiducials) present in the sample since registration is done based on ...

  • Page 379

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 373 dft fusion (discrete fourier transform fusion): the fusion is performed based upon the published method ("multiple imaging axis microscopy improves resolution for thick-sample applic...

  • Page 380

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 374 000000-2071-464 10/2014 v_01 expand to maximum volume : this checkbox is activated as default. In a multiview experiment not all views might include all the information. When expand to maximum volume is activate...

  • Page 381

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 375 5.4.19.2.2.3 use registration from file when the registration for the dataset has been determined already and the transformation matrix has been saved as an .Xml file (filename.Czi.X...

  • Page 382

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 376 000000-2071-464 10/2014 v_01 to generate the maximum intensity projection image either the front view or the side view button can be pressed. In the resulting maximum intensity projection image, each view is han...

  • Page 383

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 377 view 1 is the master view and cannot be moved during the process, the button view 1 is therefore inactive and grayed out. Any of the other views can be activated by pressing the acco...

  • Page 384

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 378 000000-2071-464 10/2014 v_01 repeat the process with the right slider and input box; here the marking lines are in blue and the illumination slider in the image control area should be placed to 2, showing the ri...

  • Page 385

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 379 ignore sample : this option activates an additional function to find structures within the sample that were incorrectly labeled as beads (fiducials). These structures are not taken i...

  • Page 386

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 380 000000-2071-464 10/2014 v_01 5.4.19.2.4 fusion after registration of the views, the fusion process can be performed (fig. 534). This will lead to one z-stack or z-stack over time. You can choose the following me...

  • Page 387

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 381 5.4.19.3 online multiview processing processing of a multiview experiment dataset can be done online during data acquisition while the data is streamed onto a storage and analysis pc...

  • Page 388

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 382 000000-2071-464 10/2014 v_01 5.4.20 processing – copy licensing instrument standard optional lsm elyra lightsheet z.1 tool accepts all types of image data as input. See full description of tool set for details. ...

  • Page 389

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 383 delete image single images or series of images can be deleted from the selected image series (input image) acquired as time series or z-stacks or combinations of those. The select bu...

  • Page 390

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 384 000000-2071-464 10/2014 v_01 5.4.21 processing – ics licensing instrument standard optional lsm elyra lightsheet z.1 tool accepts exclusively input acquired with lsm 710 and lsm 780. See full description of the ...

  • Page 391

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 385 5.4.21.2 remove structure • activate remove structures in the ics tool. The remove structures menu will appear. • to select an image, highlight the image in the image container and p...

  • Page 392

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 386 000000-2071-464 10/2014 v_01 5.4.21.3 ics correlation • activate ics correlation in the ics tool. The ics correlation menu will appear (fig. 542). • highlight the image in the image container and press the selec...

  • Page 393

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 387 5.4.21.4 map filter • activate map filter in the ics tool. The map filter menu will appear (fig. 545). • to select an image highlight the image in the image container and press the s...

  • Page 394

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 388 000000-2071-464 10/2014 v_01 5.4.21.5 nandb analysis • the function nandb (number&brightness) analysis calculates the image content in two ways: − it counts the photons per second per molecule referred to as the...

  • Page 395

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 389 − result images: number b, brightness e (molecular); this method calculates the number of molecules n and their brightness e according to the following formula: ? = (( ? ) −?????? ) ...

  • Page 396

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 390 000000-2071-464 10/2014 v_01 advanced options display negative values shifts the offset such that also negative values are displayed as a greylevel above zero. Subset length (frames) allows to define subsets of ...

  • Page 397

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 391 5.4.22 processing – adjust licensing instrument standard optional lsm elyra lightsheet z.1 * tool accepts all types of image data as input. See full description of tool set for detai...

  • Page 398

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 392 000000-2071-464 10/2014 v_01 5.4.22.2 interpolate brightness and contrast this function permits a continuous contrast and brightness adjustment in a z-stack or z-stacks over time by interpolation between the sta...

  • Page 399

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 393 5.4.22.3 channel shift channel shift is used to produce a congruent image with relation to the pixels of the various channels (fig. 550). This pixel correction function is particular...

  • Page 400

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 394 000000-2071-464 10/2014 v_01 5.4.22.4 shading correction shading correction is a tool to manipulate images which are unevenly illuminated over the field of view. This works with single images, time series, z- st...

  • Page 401

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 395 5.5 maintain tab press the maintain tab to have access to the maintain tool group area (fig. 552). The maintain tool group allow to adjust hardware settings. The maintain area is div...

  • Page 402

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 396 000000-2071-464 10/2014 v_01 5.5.1 maintain – adjust pinhole and collimators expand the adjust pinhole and collimator tab to have access to pinhole and collimator adjustment (fig. 553). This function is only ava...

  • Page 403

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 397 5.5.1.1 pinholes section panel the pinholes section panel has the following options: description field display of the relevant active channel. Diameter [µm] slider position x vis [µm...

  • Page 404

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 398 000000-2071-464 10/2014 v_01 5.5.1.2 collimators section panel the collimators section panel has the following options (fig. 555): name drop down menu selection of the collimator (ir / vis or uv / vis) to be adj...

  • Page 405

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 399 5.5.1.3 how to adjust the pinhole and collimator adjustment of pinholes can be performed manually or automatically. Temporary optimization of the adjustment: the position of the pinh...

  • Page 406

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 400 000000-2071-464 10/2014 v_01 • click the lsm continuous action button to start a continuous scan. Optimize the pinhole position in x, y and z (z only for ph1) relative to the pmt using the position x [µm] , posi...

  • Page 407

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 401 automatic pinhole adjustment: the automatic adjustment allows the pinholes to be used with any combination of beam splitters and more than one active channel. Please do not make any ...

  • Page 408

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 402 000000-2071-464 10/2014 v_01 5.5.2 maintain – objectives expand the objective tab to have access to objective definition and selection (fig. 561). With this function one can assign an objective to its respective...

  • Page 409

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 403 5.5.2.2 select objective select the objective tab from the objective tool, which will show the name of all objectives with an assigned nosepiece position . If automatic component rec...

  • Page 410

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 404 000000-2071-464 10/2014 v_01 5.5.2.3 add objective this function allows adding objectives to the user defined objective database. • click on the add objective buttons. The create new objective expansion window w...

  • Page 411

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 405 5.5.2.4 remove objective this function allows removing objectives from the user defined objective database (fig. 567). Expand the user defined objective directory and highlight the o...

  • Page 412

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 406 000000-2071-464 10/2014 v_01 5.5.2.6 focus speed press the focus speed tab to have access to the focus speed definition window (fig. 570). The focus speed can be changed for every single listed objective. Use th...

  • Page 413

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 407 for adjustment proceed as follows: position a thin sample (cell or grid) in the sample holder of the stage and focus with the first objective in the nosepiece. Press the start button...

  • Page 414

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 408 000000-2071-464 10/2014 v_01 5.5.4 maintain – airyscan the airyscan tool provides options for automatic alignment of the airyscan detector. As a superresolution detector the alignment within the detection beam p...

  • Page 415

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 409 5.5.5 options – system options the system options tool allows setting hardware parameters. Expand the tool to have access to the system options setting window (fig. 577). The left an...

  • Page 416

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 410 000000-2071-464 10/2014 v_01 5.5.5.1 system options – load configurations press the load configurations tab to open the load configurations panel (fig. 578). Load configuration lists parameters that, when their ...

  • Page 417

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 411 5.5.5.3 system options – hardware press the hardware tab to open the hardware panel (fig. 580). Hardware lists parameters that, when their respective check box is ticked, will set se...

  • Page 418

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 412 000000-2071-464 10/2014 v_01 5.5.5.4 system options – image display press the image display tab to open the image display panel (fig. 581). If the apply brightness and contrast changes to diagrams and tables is ...

  • Page 419

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 413 5.5.6 options – fcs options open the fcs options tool (fig. 582). The tool has three tabs: − data, − measure, − auto save. 5.5.6.1 fcs options – data saving raw data during measureme...

  • Page 420

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 414 000000-2071-464 10/2014 v_01 the names of the raw data files are identical to the .Fcs measurements, when stored along with them plus an extension that characterizes the measurement. Temporarily stored raw data ...

  • Page 421

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 415 the recorded word has the following structure (fig. 584): fig. 584 structure of the recorded word two examples should make the format clear (fig. 585). The tables show part of the ru...

  • Page 422

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 416 000000-2071-464 10/2014 v_01 example 1 - the following words will be recorded: − at w1: high byte: 00011001(bin) = 19 (hex); low byte: 123 (dec) = 7b (hex); resulting word = 7b19 (hex) − at w2: high byte: 000000...

  • Page 423

    Chapter 1 - system operation lsm 880 left tool area and hardware control tools zeiss 10/2014 v_01 000000-2071-464 417 5.5.6.2 fcs options – measure move stage during measure if the move stage during measure check box is activated, the stage will move during a measure- ment (fig. 587). This option al...

  • Page 424

    Chapter 1 - system operation zeiss left tool area and hardware control tools lsm 880 418 000000-2071-464 10/2014 v_01 enable laser shutter filter wheel if additional shutters (before the hene lasers and dssp lasers and a line suppression filter wheel for the argon laser) are available in the laser m...

  • Page 425: Analysis

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 419 6 center screen area / image containers - display and image analysis 6.1 structure and functional concept of the center screen area and the image display con...

  • Page 426

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 420 000000-2071-464 10/2014 v_01 fig. 591 image display window – image tabs show options view tabs: the view tabs make all viewing options and image analysis functions directly available fro...

  • Page 427

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 421 view option control tabs: these tabs allow individual activation / deactivation of the available view option control blocks by clicking on the tabs. Availabl...

  • Page 428

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 422 000000-2071-464 10/2014 v_01 in order to reset the tab layout – use the entry reset tab layout in the context menu or click on the triangle to access the view options area menu. Show all...

  • Page 429

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 423 expose mode: clicking the expose mode button in the top right corner of the image display opens a field of thumbnails in the image display with one large thu...

  • Page 430

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 424 000000-2071-464 10/2014 v_01 6.2 2d view this function allows to − display a single image in frame mode, − display multiple channel images in superimposed mode. The following view option...

  • Page 431

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 425 the functions of the zoom and tools lines are: zoom normal : the image is fitted automatically to size of the image display window which is always the origin...

  • Page 432

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 426 000000-2071-464 10/2014 v_01 (3) dimensions – reuse clicking the button transfers all acquisition parameters (exception: objective and collimator, see below) from the stored image data t...

  • Page 433

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 427 (5) dimensions – positions when clicking positions a crosshair will be displayed at the cursor position in the image. With a mouse click the position of the ...

  • Page 434

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 428 000000-2071-464 10/2014 v_01 6.2.3 player this function allows to − animate frames of a z-stack or a time series − specify animation parameters such as range and animation speed when the...

  • Page 435

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 429 6.2.4 graphics this function allows to − select from a set of drawing functions such as rectangles and arrows, − add a scale bar to the image, as well as tex...

  • Page 436

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 430 000000-2071-464 10/2014 v_01 the following list describes the most common functions in the graphics view control block. More can be found in the pull down menu. Select tool : activation ...

  • Page 437

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 431 line thickness pull down : sets the line thickness of the selected element. Color selection pull down : in this pull down, different colors can be assigned t...

  • Page 438

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 432 000000-2071-464 10/2014 v_01 to insert a coordinate label in the image display, click the button in the more … dialog. The coordinate settings dialog (fig. 608) offers the available sett...

  • Page 439

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 433 6.3 split view this function allows to − display the individual channels of a multi channel image as well as the superimposed image. The settings of the dime...

  • Page 440

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 434 000000-2071-464 10/2014 v_01 in the elyra and lsm 7 elyra systems channels of different imaging modes can be combined. For example, if sim (sr channel) or palm (hr channel) images are co...

  • Page 441

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 435 if group channels is selected all channels belonging to the same color will be merged in one image (fig. 613). If group modes is selected all channels belong...

  • Page 442

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 436 000000-2071-464 10/2014 v_01 6.4 gallery view this function allows to: − display images (z-stack, time series, combination of both) side by side in a tiled fashion, − add data relevant t...

  • Page 443

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 437 6.5 airyscan view when data are acquired with airyscan, the airyscan view tab becomes available. 6.5.1 airyscan – view option tab dimensions all airyscan dis...

  • Page 444

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 438 000000-2071-464 10/2014 v_01 6.5.2 airyscan – view option tab display raw data and sr preview due to the nature of the airyscan detection and the signal reassignment to the respective ai...

  • Page 445

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 439 6.5.3 airyscan – view option tab airyscan 2d processing image preview there are several options available for the display of the airyscan data. Source image ...

  • Page 446

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 440 000000-2071-464 10/2014 v_01 detector view shows the intensity distribution of the detection elements. This also allows a judgment whether the system is adjusted correctly (see section m...

  • Page 447

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 441 slightly reducing the sr filter strength will result in an optimal result (fig. 625). Typically, this position is 0.3 to 0.5 units higher than the auto defau...

  • Page 448

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 442 000000-2071-464 10/2014 v_01 parameters o(r): object, d(r): image, n(r): noise, eh(r): psfeff, o( ω ): object spectrum, d( ω ): image spectrum, n( ω ): noise spectrum, eh( ω ): otfeff, i...

  • Page 449

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 443 6.6 ortho view this function allows to − display a z-stack of images in an orthogonal view − measure distances in three dimensions the settings of the dimens...

  • Page 450

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 444 000000-2071-464 10/2014 v_01 6.6.1 ortho – select function • by changing the parameters x, y and z in the ortho view option control block, the section plane can be positioned at any xyz ...

  • Page 451

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 445 6.6.2 ortho – distance function • activating the tick box permits length measurements in 3d space. • click on the button to set the first xyz-point for the m...

  • Page 452

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 446 000000-2071-464 10/2014 v_01 6.7 cut view this function allows to display a user defined section plane (= cut plane) of a z-stack. It automatically improves the image of the section plan...

  • Page 453

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 447 6.8 2.5 d view this function allows to − display the two-dimensional intensity distribution of an image in an pseudo 3d mode, − show the intensity values in ...

  • Page 454

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 448 000000-2071-464 10/2014 v_01 fig. 633 2.5 d image display the 2.5 d view control tab contains the following function elements: profile button profile display (vertical polygon display). ...

  • Page 455

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 449 6.9 3d view use this view tab to display image z-stacks in three dimensions. Display and rendering of z-stacks with up to eight color channels are supported ...

  • Page 456

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 450 000000-2071-464 10/2014 v_01 3d: on the 3d view control panel the different render modes are accessible: shadow , transparent , maximum , surface and mixed . The rendering quality can be...

  • Page 457

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 451 series: 3d data can be visualized online by simply grabbing and turning the data stack with the mouse. In addition to this interactive way of interacting wit...

  • Page 458

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 452 000000-2071-464 10/2014 v_01 the zoom wheel to the left of the image display window allows continuous zooming of the 3d reconstructed image. A click on the any view button displays the 3...

  • Page 459

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 453 the following additional buttons are available in the any view shadow projection mode: • after activation of the frame button (below the image), a bounding b...

  • Page 460

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 454 000000-2071-464 10/2014 v_01 6.9.2 transparency render mode principle: in the transparent mode a three-dimensional image with a transparent effect is calculated. At least two two-dimensi...

  • Page 461

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 455 by means of the depth coding function (fig. 641), the z-information contained in a sequence can be colored with the colors of the rainbow, in which case "blu...

  • Page 462

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 456 000000-2071-464 10/2014 v_01 basic button surface rendering (voxel based) advanced button surface rendering (triangle based) full resolution button high accuracy surface rendering (trian...

  • Page 463

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 457 6.9.6 clipping planes fig. 645 image display window, 3d display, clipping planes 3d visart can display up to three so called clipping planes . These planes c...

  • Page 464

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 458 000000-2071-464 10/2014 v_01 all these functions can be used for creating a render series in order to show them and to make spectacular movies for presentations. There are three clipping...

  • Page 465

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 459 these adjustments can be made individually for each plane. The clipping planes can be chosen by selecting the respective button. Appearance of the clipping p...

  • Page 466

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 460 000000-2071-464 10/2014 v_01 clipping planes settings activate or deactivate the clipping functionality globally by the check box enable clipping . Displays the three clipping planes in ...

  • Page 467

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 461 6.9.7 flying mode the flying mode is an interactive way to explore three-dimensional data. By pressing the mouse wheel you can fly into and through a dataset...

  • Page 468

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 462 000000-2071-464 10/2014 v_01 − roughness (for shadow mode only): you can also influence the roughness used to display the surface of the image structures. This changes the plasticity of ...

  • Page 469

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 463 6.9.9 series the series tab (fig. 654) displays the render series control panel. This panel allows settings for the axis to be used for rotation of the 3d re...

  • Page 470

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 464 000000-2071-464 10/2014 v_01 (3) position list mode in the position list mode, the image is reconstructed between any required number of interim positions to be determined individually. ...

  • Page 471

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 465 6.9.10 interactive measurements 3d visart features three tools for interactive measurements in euclidian space. Measurements can be performed in all graphic-...

  • Page 472

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 466 000000-2071-464 10/2014 v_01 6.9.11 settings flying mode ticking the tick box flying mode activates the mode, unticking deactivates it. To show or hide an overview image tick the show ov...

  • Page 473

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 467 6.9.12 options xy textures only : instead of working with three texture stacks per dataset in transparent render mode, the system will only use one texture s...

  • Page 474

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 468 000000-2071-464 10/2014 v_01 intensity alpha textures : this feature optimizes the usage of the graphic ram with single channel images. Display lists activates a batch processing of the ...

  • Page 475

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 469 6.10 histogram view the histogram view ( histo view ) allows to − display a histogram (distribution of pixel intensities) of an image or region of interest, ...

  • Page 476

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 470 000000-2071-464 10/2014 v_01 fig. 666 image display, histogram view the histo button can also be used online during scanning..

  • Page 477

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 471 histogram functions: skip black and skip white tick boxes: ignore black pixels (gray value 0) and ignore white pixels (gray value 255 or 4096) in the histogr...

  • Page 478

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 472 000000-2071-464 10/2014 v_01 histogram threshold: when the threshold tick box is selected, the controls to set a lower and upper threshold for the histogram calculation are displayed (fi...

  • Page 479

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 473 fig. 669 image display, colocalization view fig. 670 scatter diagram and threshold with crosshair.

  • Page 480

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 474 000000-2071-464 10/2014 v_01 6.11.1 how a scatter diagram is generated all pixels having the same positions in both images are considered a pair. Of every pair of pixels (p1, p2) from th...

  • Page 481

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 475 6.11.2 quantitative colocalization parameters shown in the data table − no. Of pixels in image roi or scatter region − area / relative area of image roi or s...

  • Page 482

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 476 000000-2071-464 10/2014 v_01 6.11.4 overlap coefficient, overlap coefficient after manders (manders, verbeek and aten, j. Microscopy 169:375-382, 1993) ∑ ∑ ∑ = i i i i i i i ch ch ch ch ...

  • Page 483

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 477 fig. 672 image display - profile view, line profile with markers fig. 673 image display - profile view, profile displayed in image.

  • Page 484

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 478 000000-2071-464 10/2014 v_01 control block tools the profile toolbar contains the following tools: select button: activates the mouse button for selection, resizing or movement of the pr...

  • Page 485

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 479 6.13 shuttle and find view within the s&f control tab of the shuttle and find view (fig. 674) regions of interests as well as points of interest can be defin...

  • Page 486

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 480 000000-2071-464 10/2014 v_01 regions and find functions button description select by clicking on this button you activate the selection mode (button is marked blue) to select the rois or...

  • Page 487

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 481 graphical elements list list columns description eye symbol shows or hides the roi / poi in the image. Lock symbol locks a roi / poi to prevent changes. Type...

  • Page 488

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 482 000000-2071-464 10/2014 v_01 6.14 mean of roi: additional view type for time series the mean of roi view type allows to − display the intensity - time diagram (mean intensity in user def...

  • Page 489

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 483 fig. 676 image display window, mean roi display for time series in single plane fig. 677 image display window, mean roi display for time series of z-stack.

  • Page 490

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 484 000000-2071-464 10/2014 v_01 the mean of roi view options control block contains the following function elements: rois can be managed in the graphics view option control block. Arrow but...

  • Page 491

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 485 buttons for diagram display options: mode pull down: choose between area and mean mode area: display of the area of the roi in the intensity time diagram, de...

  • Page 492

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 486 000000-2071-464 10/2014 v_01 6.15 kinetic / frap view: additional view type for time series the frap function permits interactive analysis of bleaching experiments, including − correctio...

  • Page 493

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 487 available tools in the frap view: channels select single channels or all channels for analysis in the general dimensions view options control box. Kinetic mo...

  • Page 494

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 488 000000-2071-464 10/2014 v_01 example: frap performed in a nucleus expressing gfp labeled proteins display of the image series in the mean roi display mode: the drawing tools are used to ...

  • Page 495

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 489 • switch to the frap view tab. • mark the check boxes for background and reference region. • the intensity values of the roi to be analyzed will be corrected...

  • Page 496

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 490 000000-2071-464 10/2014 v_01 fig. 681 image window displaying the analysis of frap data using a mono exponential fit fig. 682 image window displaying the analysis of frap data using a do...

  • Page 497

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 491 if the analysis is done using the double exponential fit the fitted curve displays the mean of the fitted values for the two different mobile fractions. The ...

  • Page 498

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 492 000000-2071-464 10/2014 v_01 6.16 lambda coded: additional view types for lambda mode the lambda coded view displays a lambda stack in a wavelength-coded color view. A color palette is a...

  • Page 499

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 493 fig. 685 dimensions view option control block for lambda coded view.

  • Page 500

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 494 000000-2071-464 10/2014 v_01 6.17 unmixing view in the unmixing view , lambda or channel data sets can be analyzed and spectrally unmixed (for more information see as well section proces...

  • Page 501

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 495 fig. 688 unmixing display of a lambda stack acquired on an lsm 710 / lsm 780

  • Page 502

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 496 000000-2071-464 10/2014 v_01 fig. 689 unmixing display of a lambda stack with a spectral dataset acquired on an lsm 700 with a variable secondary dichroic.

  • Page 503

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 497 tools in the unmixing view options control block − tools for display customization and drawing: drawing tools work the same as in the graphics view option co...

  • Page 504

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 498 000000-2071-464 10/2014 v_01 an additional channel per unmixed component is displayed which shows the relative standard deviation (rsd) in each unmixed component channel (see also sectio...

  • Page 505

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 499 6.17.1 automatic component extraction the use of this function permits the automatic search for the individual reference spectra in a lambda stack. • load or...

  • Page 506

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 500 000000-2071-464 10/2014 v_01 tools in the unmixing - extract view options control block: these tools (unmixing – extract) are only available for lambda stacks on an lsm 710 and lsm 780 s...

  • Page 507

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 501 6.18 fret view in the fret view type (fig. 693), fret data sets can be analyzed: − data gained performing acceptor photobleaching − data gained performing se...

  • Page 508

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 502 000000-2071-464 10/2014 v_01 fretn (p) : result of analysis using gordon method. The fretn value is calculated for each pixel, afterwards the average for the chosen region is determined....

  • Page 509

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 503 6.18.1 tools in the fret view options control block for acceptor photobleaching fret tab (fig. 695): as method for fret analysis, acceptor photobleach is aut...

  • Page 510

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 504 000000-2071-464 10/2014 v_01 thresholds tab (fig. 697): the threshold for the image analysis can be set manually using the slider or editing text box next to donor , acceptor or all , wh...

  • Page 511

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 505 6.18.2 tools in the fret view options control block for sensitized emission fret tab (fig. 700): within this tab overlay regions can be defined. (the drawing...

  • Page 512

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 506 000000-2071-464 10/2014 v_01 n-fret or xia method: xia and liu, biophys j 81 , 2395 (2001) nfret = fret 1/ √ (dfd*afa) displays an n-fret image with intensities converted from the fret i...

  • Page 513

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 507 thresholds tab (fig. 702): the threshold for the image analysis can be set manually using the slider or editing text box next to donor , acceptor , fret or a...

  • Page 514

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 508 000000-2071-464 10/2014 v_01 6.19 raster scanning image correlation spectroscopy (rics) rics is an image analysis tool that allows retrieving dynamic information from intensity images or...

  • Page 515

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 509 6.19.1 access to rics if you have taken an image or an image series or have opened an old image or image stack, a click on the rics tab of the view tab list ...

  • Page 516

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 510 000000-2071-464 10/2014 v_01 6.19.3 correlation functionalities activate correlation under the rics view. The correlation options will be displayed in the view control window (fig. 705)....

  • Page 517

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 511 fig. 706 image window with two selected regions of interest in the intensity image correlation allows you to select the type of correlation analysis you want...

  • Page 518

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 512 000000-2071-464 10/2014 v_01 for a two channel cross-correlation setup, two drop-down menus will be displayed (fig. 709). The upper channel will be the one that will be cross- correlated...

  • Page 519

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 513 when immobile structures (+stack) is selected, the average image from the stack (pixel by pixel) is subtracted from every image and a scalar is added to avoi...

  • Page 520

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 514 000000-2071-464 10/2014 v_01 note, that for different representations different options will become available. For the diagram , table and 2.5d display , the pixel pull down menu becomes...

  • Page 521

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 515 for the diagram and table, the x and y pull down menu and the draw symmetric curve check box will be additionally available. In the x and y pull down menu yo...

  • Page 522

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 516 000000-2071-464 10/2014 v_01 checking and unchecking the show fit-results box will toggle between the correlation and fit images. Note, if this box is activated, the interpolate g(0) box...

  • Page 523

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 517 if you press the settings button, the settings menu will pop up (fig. 723). In map selection you can determine the map type with the map type pull down menu ...

  • Page 524

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 518 000000-2071-464 10/2014 v_01 in the show all mode you have further the option to display the std.Dev. (standard deviation) maps of both parameter maps as well as the Χ 2 (chi square) map...

  • Page 525

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 519 the map size in terms of region numbers is displayed in the info area of the settings menu. By pressing generate a map is created with region and shift sizes...

  • Page 526

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 520 000000-2071-464 10/2014 v_01 6.19.5 fitting functionalities activate fitting under the rics view. The fit options will be displayed in the view control window (fig. 727). Fig. 727 fit co...

  • Page 527

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 521 you can select a model from the model pull down menu in the fit table. Every stored model will be listed (fig. 729). Below the model pull down menu the activ...

  • Page 528

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 522 000000-2071-464 10/2014 v_01 by clicking with the right mouse button into the result table the table’s menu box will pop up (fig. 733). You have the option to copy the table to the clipb...

  • Page 529

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 523 6.19.6 model functionalities activate model under the rics view. The model options will be displayed in the view control window (fig. 736). Fig. 736 fit cont...

  • Page 530

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 524 000000-2071-464 10/2014 v_01 for three of the terms (g s , a and g d ) settings menus are available. To open a settings menu, press the corresponding settings button. Scanning g s ( ξ , ...

  • Page 531

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 525 translation g d ( ξ , ψ ) describes the translational diffusion term of the model function. To open the translation settings press the settings button (fig. ...

  • Page 532

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 526 000000-2071-464 10/2014 v_01 components allow selecting 1 , 2 or 3 components by pressing the respective button. Free / anomalous allow via pull- down menus to select between free transl...

  • Page 533

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 527 6.20 polarization imaging linear (plane) polarized light, which is light whose wave goes only one direction, exciting a fluorescent molecule with a preferred...

  • Page 534

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 528 000000-2071-464 10/2014 v_01 6.20.1 imaging setup for polarization imaging in case emission polarizers (analyzers) are available, you have a notch filter cascade selection button availab...

  • Page 535

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 529 6.20.2 determination of g-factor in order to determine the g-factor for the lsm 710 and lsm 780 systems, you need an isotropic medium like a fluorescent dye ...

  • Page 536

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 530 000000-2071-464 10/2014 v_01 6.20.3 obtaining an anisotropy image set up a multitrack configuration with alternating analyzers: p in track 1, s in track 2, leaving all other settings equ...

  • Page 537

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 531 in the ratio1 channel choose the ratio type 3 formula. With ch1-t1 and ch1-t2 corresponding to i p and i s this formula corresponds to the formula for anisot...

  • Page 538

    Chapter 1 - system operation zeiss center screen area / image containers - display and image analysis lsm 880 532 000000-2071-464 10/2014 v_01 6.21 information view the information view is a view type that shows a summary information sheet of all relevant image acquisition parameters. Parts of it ca...

  • Page 539

    Chapter 1 - system operation lsm 880 center screen area / image containers - display and image analysis zeiss 10/2014 v_01 000000-2071-464 533 fig. 751 information view of a processed superresolution image.

  • Page 540

    Chapter 1 - system operation zeiss right tool area, data management and storage lsm 880 534 000000-2071-464 10/2014 v_01 7 right tool area, data management and storage 7.1 general document type images are stored as files (*.Czi format) in folders just like in any other windows application. All infor...

  • Page 541

    Chapter 1 - system operation lsm 880 right tool area, data management and storage zeiss 10/2014 v_01 000000-2071-464 535 7.2 zen file browser the zen file browser is a versatile tool to access your image data. • in the menu bar, click on file and select the file browser … item to open the zen file b...

  • Page 542

    Chapter 1 - system operation zeiss right tool area, data management and storage lsm 880 536 000000-2071-464 10/2014 v_01 eventual info messages (fig. 753) fading in the right hand corner inform about missing hardware if applicable. The message box disappears after ~ 10 seconds. The images can be sor...

  • Page 543

    Chapter 1 - system operation lsm 880 right tool area, data management and storage zeiss 10/2014 v_01 000000-2071-464 537 within the dialog (fig. 756) use the editing boxes or the sliders to set the desired values. The slider box toggles with the highlighted dimension. Click select file name and save...

  • Page 544

    Chapter 1 - system operation zeiss right tool area, data management and storage lsm 880 538 000000-2071-464 10/2014 v_01 7.2.1 gallery view of the zen file browser in the gallery view of the browser the images are displayed as thumbnails. One click selects a single image, press shift and click to se...

  • Page 545

    Chapter 1 - system operation lsm 880 right tool area, data management and storage zeiss 10/2014 v_01 000000-2071-464 539 fig. 759 images with information.

  • Page 546

    Chapter 1 - system operation zeiss right tool area, data management and storage lsm 880 540 000000-2071-464 10/2014 v_01 7.2.2 form view of the zen file browser all images in a given folder can be accessed by using the slider below the info-panel or using the stepper on the right side of the slider....

  • Page 547

    Chapter 1 - system operation lsm 880 right tool area, data management and storage zeiss 10/2014 v_01 000000-2071-464 541 7.2.3 table view of the zen file browser the table view is especially useful in case a lot of files are found in one folder. The columns displayed in the table are selectable from...

  • Page 548

    Chapter 1 - system operation zeiss right tool area, data management and storage lsm 880 542 000000-2071-464 10/2014 v_01 7.3 images and documents panel the images and documents panel (fig. 763) is located in the right tool area of zen. It displays all images and data which are open in zen. Its purpo...

  • Page 549

    Chapter 1 - system operation lsm 880 right tool area, data management and storage zeiss 10/2014 v_01 000000-2071-464 543 the context menu of the images and documents section allows the following functions: − selecting one of the view options textual view , thumbnail view or big view − selecting all ...

  • Page 550

    Chapter 1 - system operation zeiss right tool area, data management and storage lsm 880 544 000000-2071-464 10/2014 v_01 − a progress bar (fig. 767) is shown for each image facilitating the overview if e.G. Many processing function are running at the same time. − in case long filenames are used the ...

  • Page 551

    Chapter 1 - system operation lsm 880 right tool area, data management and storage zeiss 10/2014 v_01 000000-2071-464 545 7.4 opening of files via the "open" command in the file menu • click on the file menu of the menu bar and select open or click on the icon of the main toolbar. − the standard wind...

  • Page 552

    Chapter 1 - system operation zeiss right tool area, data management and storage lsm 880 546 000000-2071-464 10/2014 v_01 7.6 export of images the export function allows the export of acquired images and images loaded. • select the image to be exported. • click on the export menu item in the file men...

  • Page 553: Index

    Chapter 1 - system operation lsm 880 index zeiss 10/2014 v_01 000000-2071-464 547 8 index 2 2d view ...................................................... 424 3 32 channel module light path ...................... 183 3d distance measurement ............................ 445 3d view .....................

  • Page 554

    Chapter 1 - system operation zeiss index lsm 880 548 000000-2071-464 10/2014 v_01 subset ............................................ 382, 383 copy channel .............................................. 382 correction ................................................... 148 correlation .................

  • Page 555

    Chapter 1 - system operation lsm 880 index zeiss 10/2014 v_01 000000-2071-464 549 image tabs .................................................... 46 image view controls ....................................... 46 image view tabs ............................................ 46 images and documents pan...

  • Page 556

    Chapter 1 - system operation zeiss index lsm 880 550 000000-2071-464 10/2014 v_01 sample carrier ......................................... 229 stage ..................................................... 228 ndd light path ............................................ 181 nearest neighbour ..............

  • Page 557

    Chapter 1 - system operation lsm 880 index zeiss 10/2014 v_01 000000-2071-464 551 acquisition .............................................. 190 correlator ............................................... 224 fit ........................................................... 192 imaging setup..............

  • Page 558

    Chapter 1 - system operation zeiss index lsm 880 552 000000-2071-464 10/2014 v_01 zoom function .................................... 275, 284 z-piezo ........................................................ 146 z-stack ........................................................ 141 z-stack settings for...

  • Page 559: Chapter 2

    Chapter 2 - macros and visual basic lsm 880 contents zeiss 10/2014 v_01 000000-2071-464 1 chapter 2 macros and visual basic contents page 1 vba version ........................................................................................................... 3 2 macros ................................

  • Page 560

    Chapter 2 - macros and visual basic zeiss contents lsm 880 2 000000-2071-464 10/2014 v_01 4.8 example macros ................................................................................................................ 33 4.8.1 access to images .......................................................

  • Page 561: Vba Version

    Chapter 2 - macros and visual basic lsm 880 vba version zeiss 10/2014 v_01 000000-2071-464 3 1 vba version both versions of vba (visual basic for applications) – version 6.4 (32 bit) and version 7.1 (64 bit) – are available. Version 6.4 is registered by default to ensure compatibility to existing ma...

  • Page 562

    Chapter 2 - macros and visual basic zeiss macros lsm 880 4 000000-2071-464 10/2014 v_01 the vba functionality described in this paragraph refers to vba as implemented in the zeiss lsm software 4.2. With the introduction of zen, the range of functions is and will be extended, while vba can still be u...

  • Page 563

    Chapter 2 - macros and visual basic lsm 880 macros zeiss 10/2014 v_01 000000-2071-464 5 macros are stored and managed in project files (*.Lvb). Before you can record or edit a macro, you have to create a project as follows: • press the new button to create a project file (see fig. 4). − a new projec...

  • Page 564

    Chapter 2 - macros and visual basic zeiss macros lsm 880 6 000000-2071-464 10/2014 v_01 (2) recording panel before recording a command sequence, you can enter the name for the macro to be created in the name input box of the recording panel. Start button: starts recording. Cancel button: cancels the...

  • Page 565

    Chapter 2 - macros and visual basic lsm 880 macros zeiss 10/2014 v_01 000000-2071-464 7 proceed as follows to edit a macro: • select the required macro from the project list box of the macros panel. • click on the edit button. The microsoft visual basic editing window will be opened. • make the requ...

  • Page 566

    Chapter 2 - macros and visual basic zeiss macros lsm 880 8 000000-2071-464 10/2014 v_01 2.2.3 editing and debugging of macros the edit in the edit macro panel button activates ide (integrated development environment), which allows macros to be edited and debugged. Under the help - macrosoft visual b...

  • Page 567

    Chapter 2 - macros and visual basic lsm 880 macros zeiss 10/2014 v_01 000000-2071-464 9 2.3 overview of available macros documentation files (*.Rtf, *.Doc) of advanced macros are located in the macro directory. Additional macros for service are listed in a separate folder. Name description cameracol...

  • Page 568

    Chapter 2 - macros and visual basic zeiss macros lsm 880 10 000000-2071-464 10/2014 v_01 name description sphericobjectivecorrection60.Lvb calibrates the spheric component of objective error by automatic acquisition of a z-stack on a flat surface and a spherical fit of the topographic data. Testgrid...

  • Page 569

    Chapter 2 - macros and visual basic lsm 880 macros zeiss 10/2014 v_01 000000-2071-464 11 2.5 excitationfingerprinting with tunable multiphoton lasers (except opo devices) it is possible to acquire excitation spectra and subsequently use these spectra to perform linear unmixing. This process is calle...

  • Page 570

    Chapter 2 - macros and visual basic zeiss macros lsm 880 12 000000-2071-464 10/2014 v_01 1) chose the aom calibration tab. 2) set aom transmission to a value around 10. 3) set the laser to the lowest wavelength possible (ideally this has already been done in the main software). 4) start a continuous...

  • Page 571

    Chapter 2 - macros and visual basic lsm 880 macros zeiss 10/2014 v_01 000000-2071-464 13 the normalized aom values will provide the right aom settings starting with the chosen setting at a given wavelength and calculating the relative values for all other wavelengths. Thus it is avoided to reach max...

  • Page 572

    Chapter 2 - macros and visual basic zeiss macros lsm 880 14 000000-2071-464 10/2014 v_01 2.6 concatenateimages the mcacro allows concatenating various types of images into one image document showing the final image as a time series image. It is useful to combine images from multi block experiments i...

  • Page 573: Visual Macro Editor

    Chapter 2 - macros and visual basic lsm 880 visual macro editor zeiss 10/2014 v_01 000000-2071-464 15 3 visual macro editor the visual macro editor is an optional software module which allows the user to program a variety of scanning procedures, image calculations or image processing routines as wel...

  • Page 574

    Chapter 2 - macros and visual basic zeiss visual macro editor lsm 880 16 000000-2071-464 10/2014 v_01 3.2 function description toolbar buttons run starts the macro starting with the first block in the program flow column. Stop stops the macro at any time. Break interrupts the macro. Clicking the but...

  • Page 575

    Chapter 2 - macros and visual basic lsm 880 visual macro editor zeiss 10/2014 v_01 000000-2071-464 17 • click at the action block with the left mouse button (hold down the mouse button) and drag the mouse cursor into the flow column. Release the mouse button. − action blocks for the program flow sho...

  • Page 576

    Chapter 2 - macros and visual basic zeiss visual macro editor lsm 880 18 000000-2071-464 10/2014 v_01 acquisition action blocks loads a beam path configuration form the configuration list or uses the current one that is set up in the main program. Use the pull down menu in the property/value list to...

  • Page 577

    Chapter 2 - macros and visual basic lsm 880 visual macro editor zeiss 10/2014 v_01 000000-2071-464 19 this block performs the find function according to the main software. This action block sets the z-position according to the focusing procedure set with the parameters available. The image generated...

  • Page 578

    Chapter 2 - macros and visual basic zeiss visual macro editor lsm 880 20 000000-2071-464 10/2014 v_01 it is possible to export images choosing a certain file format from the pull down list that appears when highlighting the line file format in the properties list. Specific parameters are assigned to...

  • Page 579

    Chapter 2 - macros and visual basic lsm 880 visual macro editor zeiss 10/2014 v_01 000000-2071-464 21 process action blocks this block allows to do image calculation using the pixel intensities of the image. The operators for the formulas that can be used are described in the ratio dialogue within t...

  • Page 580

    Chapter 2 - macros and visual basic zeiss visual macro editor lsm 880 22 000000-2071-464 10/2014 v_01 this action block defines a mask (image region with defined properties) derived from an image window. The image can be a newly acquired or imported image. The properties of the mask based on the ima...

  • Page 581

    Chapter 2 - macros and visual basic lsm 880 visual macro editor zeiss 10/2014 v_01 000000-2071-464 23 sub macro actions blocks this block allows setting a sub macro within a main macro. The sup macro is opened by clicking onto the arrow in the block icon and closed clicking the top right of the prog...

  • Page 582

    Chapter 2 - macros and visual basic zeiss visual macro editor lsm 880 24 000000-2071-464 10/2014 v_01 • assign the value 1 to a variable named index (see fig. 13). • click the single action blocks and assign the values to the listed properties (beam path and scan parameters can be chosen according t...

  • Page 583

    Chapter 2 - macros and visual basic lsm 880 visual macro editor zeiss 10/2014 v_01 000000-2071-464 25 • the movement of the stage is defined highlighting the move stage action block, chosing the mode relative position in the property list and typing in the value 10 for x and y which then will move t...

  • Page 584: Vba Programming For Lsm

    Chapter 2 - macros and visual basic zeiss vba programming for lsm lsm 880 26 000000-2071-464 10/2014 v_01 4 vba programming for lsm 4.1 vba programming visual basic for applications (vba) is a widely-spread programming language, and lots of sources for an introduction to the language can be found on...

  • Page 585

    Chapter 2 - macros and visual basic lsm 880 vba programming for lsm zeiss 10/2014 v_01 000000-2071-464 27 4.3 object browser to search the structure of an api object, use the object browser from the vba editor. To explore an object from your vba code, position the curser in the object and press "f2"...

  • Page 586

    Chapter 2 - macros and visual basic zeiss vba programming for lsm lsm 880 28 000000-2071-464 10/2014 v_01 4.4 com interface the full range of zen vba commands is also available via com, by connecting a client program to a running zen system. There are two com-automation objects available, for the ls...

  • Page 587

    Chapter 2 - macros and visual basic lsm 880 vba programming for lsm zeiss 10/2014 v_01 000000-2071-464 29 application or lsm5application lsm5 dsrecordingdoc lsm5hardware lsm5info lsm5tools lsm5vbaproject lsm5constants lsm5options cpamplifiers cpcollimators cpfiltersets cpfocus cpintegrators cplamps ...

  • Page 588

    Chapter 2 - macros and visual basic zeiss vba programming for lsm lsm 880 30 000000-2071-464 10/2014 v_01 4.7 programming with the vba-gui interface 4.7.1 introduction the zen vba-gui interface allows you to program zen with the same comfort and ease as using the mouse to control the gui. All capabi...

  • Page 589

    Chapter 2 - macros and visual basic lsm 880 vba programming for lsm zeiss 10/2014 v_01 000000-2071-464 31 4.7.2 structure of a vba-gui command there are three different kinds of actions that can be performed in the zen gui: setting a value (either a number or a string, e.G. The frame size or configu...

  • Page 590

    Chapter 2 - macros and visual basic zeiss vba programming for lsm lsm 880 32 000000-2071-464 10/2014 v_01 4.7.4 selecting and managing image documents image document handling in the vba-gui interface happens via the "gui.Document" command. From the list of open documents, images can be selected by i...

  • Page 591

    Chapter 2 - macros and visual basic lsm 880 vba programming for lsm zeiss 10/2014 v_01 000000-2071-464 33 4.8 example macros this library of example macros is designed as a starting point for your own programming ventures. They are deliberately kept simple for quick understanding and hand-on. We rec...

  • Page 592

    Chapter 2 - macros and visual basic zeiss vba programming for lsm lsm 880 34 000000-2071-464 10/2014 v_01 4.8.5 vba-gui api we have not created example macros for the vba-gui api, because these can easily be generated by the macro recorder functionality. Activate macro recording and the execute the ...

  • Page 593: Chapter 3

    Chapter 3 - additional software tools lsm 880 contents zeiss 10/2014 v_01 000000-2071-464 1 chapter 3 additional software tools contents page 1 stand select ........................................................................................................... 2 2 systemmaintenance tool ...........

  • Page 594: Stand Select

    Chapter 3 - additional software tools zeiss stand select lsm 880 2 000000-2071-464 10/2014 v_01 1 stand select the stand select tool permits a new or updated database to be assigned to the zen software program. • close the zen software program and double- click on the stand select icon on the deskto...

  • Page 595: Systemmaintenance Tool

    Chapter 3 - additional software tools lsm 880 systemmaintenance tool zeiss 10/2014 v_01 000000-2071-464 3 2 systemmaintenance tool with a calibration objective and correct system configuration, the systemmaintenance tool allows convenient self-adjustment of lsm. The optical beampath, relative pinhol...

  • Page 596

    Chapter 3 - additional software tools zeiss systemmaintenance tool lsm 880 4 000000-2071-464 10/2014 v_01 2.2 starting the maintenance tool before starting the maintenance tool, the system hardware must be fully activated. To do this, open zen and check the following settings: correct entry of all e...

  • Page 597

    Chapter 3 - additional software tools lsm 880 systemmaintenance tool zeiss 10/2014 v_01 000000-2071-464 5 the maintenance tool can only be used, if the calibration objective is entered as apo calibration lsm . If this is not the case, a message will remind the user to select the calibration objectiv...

  • Page 598

    Chapter 3 - additional software tools zeiss systemmaintenance tool lsm 880 6 000000-2071-464 10/2014 v_01 fig. 9 maintenance tool main window fig. 10 the maintenance tool guides users through required adjustment steps.

  • Page 599: Zen Configuration Tool

    Chapter 3 - additional software tools lsm 880 zen configuration tool zeiss 10/2014 v_01 000000-2071-464 7 3 zen configuration tool the configuration tool provides access to the database of a system. Be aware that wrong entries can lead to a malfunction of the system. Always keep a backup of the orig...

  • Page 600

    Chapter 3 - additional software tools zeiss zen configuration tool lsm 880 8 000000-2071-464 10/2014 v_01 • within the appearing window (fig. 12) choose the tabs scan head , lsm 880 , filter . • do not change any existing filters or filters from filterwheels inside the scan head. This leads to malfu...

  • Page 601

    Chapter 3 - additional software tools lsm 880 zen configuration tool zeiss 10/2014 v_01 000000-2071-464 9 after selecting the "filterwheel", the current filters are indicated each with a button next to it referring to the detection channel it is effective for (fig. 12). • clicking this button opens ...

  • Page 602

    Chapter 3 - additional software tools zeiss zen configuration tool lsm 880 10 000000-2071-464 10/2014 v_01 • indicate the new filter as non zeiss and fill in the form to provide all necessary information to the system so it can use the information for image acquisition and processing. The form on ho...

  • Page 603: Chapter 4

    Chapter 4 - annex lsm 880 contents zeiss 10/2014 v_01 000000-2071-464 1 chapter 4 annex contents page 1 software and hardware options ......................................................................... 2 1.1 software ................................................................................

  • Page 604

    Chapter 4 - annex zeiss software and hardware options lsm 880 2 000000-2071-464 10/2014 v_01 1 software and hardware options this section describes optional software and hardware configurations. Depending on your configuration, the content of dialogue and function may differ. 1.1 software the follow...

  • Page 605

    Chapter 4 - annex lsm 880 software and hardware options zeiss 10/2014 v_01 000000-2071-464 3 if your configuration does not include the "zen module visual basic macro, vba", the following functions are not available: − new , save and save as buttons in the macro control window − edit , step , delete...

  • Page 606

    Chapter 4 - annex zeiss software and hardware options lsm 880 4 000000-2071-464 10/2014 v_01 1.2 hardware depending on whether the following hardware components are available or not, the content of the screens may differ: − piezo objective focusing device / z-piezo stage − x-y scanning stage (differ...

  • Page 607

    Chapter 4 - annex lsm 880 courses on "how to operate the system … zeiss 10/2014 v_01 000000-2071-464 5 2 courses on "how to operate the system in an optimized way" zeiss is offering training courses on how to operate the system in an optimized way. − courses are held in our application center in jen...

  • Page 608

    Chapter 4 - annex zeiss beam path configuration guide for … lsm 880 6 000000-2071-464 10/2014 v_01 3.2 setup of tracks using single detectors (channel mode) a) one track only – simultaneous acquisition of channels • switch on the suitable lasers for excitation of the dyes in the specimen in the lase...

  • Page 609

    Chapter 4 - annex lsm 880 beam path configuration guide for … zeiss 10/2014 v_01 000000-2071-464 7 • add a new track by clicking the button next to tracks in the imaging setup tool. • create a second track for af 568. • by selecting the dyes from the pull-down menu, you can see the emission spectra ...

  • Page 610

    Chapter 4 - annex zeiss beam path configuration guide for … lsm 880 8 000000-2071-464 10/2014 v_01 line and frame mode of multitracking • settings can be used for line or frame wise multi-tracking acquisition. • in line mode the lines are scanned in turns for all tracks with the corresponding laser ...

  • Page 611: Of Dyes

    Chapter 4 - annex lsm 880 recommendations for excitation laser lines and emission filters of dyes zeiss 10/2014 v_01 000000-2071-464 9 4 recommendations for excitation laser lines and emission filters of dyes dye laser line/mbs emission bands dapi 405 > 385/420, max. At 461 ebfp 405 > 385/420, max. ...

  • Page 612

    Chapter 4 - annex zeiss how to access data acquired with airyscan using other software lsm 880 10 000000-2071-464 10/2014 v_01 5 how to access data acquired with airyscan using other software there are different ways to access the airyscan raw pixel data: − via zen black vba macro interface − czi fi...

  • Page 613: Piezo Focusing Device

    Chapter 4 - annex lsm 880 piezo focusing device zeiss 10/2014 v_01 000000-2071-464 11 6 piezo focusing device a z-piezo allows z-stacks to be acquired considerably quicker compared to the focus drive of the microscope stand. The speed advantage depends on the total travel range in z and the speed of...

  • Page 614

    Chapter 4 - annex zeiss piezo focusing device lsm 880 12 000000-2071-464 10/2014 v_01 objectives: − m27 installation: • screw in your microscope objective into piezo objective focusing device (see fig. 3/ 1 ). • screw the thread-ring into your microscope (see fig. 3/ 2 ). • easy clamp the piezo obje...

  • Page 615

    Chapter 4 - annex lsm 880 specification of trigger-interface zeiss 10/2014 v_01 000000-2071-464 13 7 specification of trigger-interface the lsm provide two ways to control external devices or be controlled via external devices. Synchronization signals: this interface is provided directly by the elec...

  • Page 616

    Chapter 4 - annex zeiss specification of trigger-interface lsm 880 14 000000-2071-464 10/2014 v_01 connectors: pixelclock = coax: triax lemo s. A. Serie 00, epl.00.650nln pin signal name description signal pclk_out pixelclock output signal shield gnd digital ground outer shield shield chassis ground...

  • Page 617

    Chapter 4 - annex lsm 880 specification of trigger-interface zeiss 10/2014 v_01 000000-2071-464 15 15-point high density d-type plug: plug "b" pin signal name description hardware output only 1 syncout4 ** (stack) synchronous output sync. Signal: stack out 2 syncout5 out grabbing synchronous output ...

  • Page 618

    Chapter 4 - annex zeiss specification of trigger-interface lsm 880 16 000000-2071-464 10/2014 v_01 type/voltage range: − ttl signal level 3.3 v, cmos low power consumption − 5.0 v tolerant input/output for interfacing with 5 v logic load: output: input: 4.7 kohm input impedance (internal 4.7 kohm pu...

  • Page 619

    Chapter 4 - annex lsm 880 specification of trigger-interface zeiss 10/2014 v_01 000000-2071-464 17 user interface box for synchronization and trigger signals (optional) the interface box provides connections to the trigger in and out functions accessible in the software user interface, the synchroni...

  • Page 620: (Elyra)

    Chapter 4 - annex zeiss level-adjustable sample holder with insert for piezo stage (elyra) lsm 880 18 000000-2071-464 10/2014 v_01 8 level-adjustable sample holder with insert for piezo stage (elyra) caution! The adjustment screws and other part of adjustable sample holders may collide with parts of...

  • Page 621

    Chapter 4 - annex lsm 880 level-adjustable sample holder with insert for piezo stage (elyra) zeiss 10/2014 v_01 000000-2071-464 19 e) press the lower pointing arrow of the x input box of the stage tool to return to the initial position. If the image is in focus (fig. 6/ f ) x leveling is complete; o...

  • Page 622

    Chapter 4 - annex zeiss level-adjustable sample holder with insert for piezo stage (elyra) lsm 880 20 000000-2071-464 10/2014 v_01 2. Draw fiber plug of x-cite completely out from the x-cite casing. Start acquisition. The image will be dark since fiber was pulled out. Put fiber back and slowly move ...

  • Page 623

    Chapter 4 - annex lsm 880 level-adjustable sample holder for lsm zeiss 10/2014 v_01 000000-2071-464 21 9 level-adjustable sample holder for lsm caution! Adjustable stage insert screws may intersect with parts of the microscope, e.G. The condenser unit. When moving a motorized stage, avoid any collis...

  • Page 624

    Chapter 4 - annex zeiss level-adjustable sample holder for lsm lsm 880 22 000000-2071-464 10/2014 v_01 3. Start a reflection scan at zoom 1.0x and focus on the border between coverslip and sample, which can be identified by the brightest reflection. A high pixel resolution is not important (use e.G....

  • Page 625

    Chapter 4 - annex lsm 880 level-adjustable sample holder for lsm zeiss 10/2014 v_01 000000-2071-464 23 3. Start a reflection bidi line bidi z scan at zoom 1.0x and focus on the border between coverslip and sample, which can be identified by the brightest reflection line. A high pixel resolution is n...

  • Page 626

    Chapter 4 - annex zeiss liquid cooling system of the lsm lsm 880 24 000000-2071-464 10/2014 v_01 10 liquid cooling system of the lsm the temperature of the scanners within the lsm scan head is controlled with a liquid cooling system. The liquid cooling system is also used to dissipate the heat gener...

  • Page 627: Chapter 5

    Chapter 5 - multiphoton laser scanning … lsm 880 contents zeiss 10/2014 v_01 000000-2071-464 1 chapter 5 multiphoton laser scanning microscopy with lsm family systems contents page 1 nlo (non linear optics) laser for lsm ............................................................... 2 1.1 laser con...

  • Page 628

    Chapter 5 - multiphoton laser scanning … zeiss nlo (non linear optics) laser for lsm lsm 880 2 000000-2071-464 10/2014 v_01 1 nlo (non linear optics) laser for lsm the lsm scanning module is operated with a titanium-sapphire-laser and optionally in combination with an optical parametric oscillator (...

  • Page 629

    Chapter 5 - multiphoton laser scanning … lsm 880 nlo (non linear optics) laser for lsm zeiss 10/2014 v_01 000000-2071-464 3 1.1 laser control tool the laser control tool shows the nlo laser as an additional laser of the system. Operation of the chameleon (coherent), opo and mai tai (spectra physics)...

  • Page 630

    Chapter 5 - multiphoton laser scanning … zeiss nlo (non linear optics) laser for lsm lsm 880 4 000000-2071-464 10/2014 v_01 1.2 imaging setup and channels tool • setting the wavelength of the laser/opo • the wavelength of the laser and opo can be set in the imaging setup tool in the pop up window of...

  • Page 631

    Chapter 5 - multiphoton laser scanning … lsm 880 nlo (non linear optics) laser for lsm zeiss 10/2014 v_01 000000-2071-464 5 1.3 motorized periscope • systems with opo or two lasers coupled into one scan head are equipped with a motorized periscope. The interface for controlling the motorized mirrors...

  • Page 632

    Chapter 5 - multiphoton laser scanning … zeiss nlo (non linear optics) laser for lsm lsm 880 6 000000-2071-464 10/2014 v_01 1.3.1 instructions for beam adjustment using the motorized periscope your system is equipped with a periscope that provides motorised mirrors for the guidance of two beams of l...

  • Page 633

    Chapter 5 - multiphoton laser scanning … lsm 880 nlo (non linear optics) laser for lsm zeiss 10/2014 v_01 000000-2071-464 7 motorized periscope panel this panel provides the interface to control the motorized mirrors in the periscope beam 1 and beam 2 represent the two input beams according to the f...

  • Page 634

    Chapter 5 - multiphoton laser scanning … zeiss nlo (non linear optics) laser for lsm lsm 880 8 000000-2071-464 10/2014 v_01 the following procedures describe how to adjust positioning and illumination for the acquired images using the zen software. 1.3.1.1 correction of the illumination profile the ...

  • Page 635

    Chapter 5 - multiphoton laser scanning … lsm 880 nlo (non linear optics) laser for lsm zeiss 10/2014 v_01 000000-2071-464 9 horizontal shift of the image generated with beam1 (nlo laser) to the right (to shift the image horizontally to the left, just use the reverse direction buttons): 1. Click on t...

  • Page 636

    Chapter 5 - multiphoton laser scanning … zeiss nlo (non linear optics) laser for lsm lsm 880 10 000000-2071-464 10/2014 v_01 vertical shift of the image generated with beam1 (nlo laser) downwards (to shift the image upwards, just use the reverse direction buttons): 1. Click the right "fine movement"...

  • Page 637

    Chapter 5 - multiphoton laser scanning … lsm 880 nlo (non linear optics) laser for lsm zeiss 10/2014 v_01 000000-2071-464 11 1.4 pinhole and collimator settings • the pinhole can be fully opened for maximum detection efficiency due to the intrinsic optical sectioning capabilities of the nlo effect (...

  • Page 638: Non Descanned Detection

    Chapter 5 - multiphoton laser scanning … zeiss non descanned detection (ndd) lsm 880 12 000000-2071-464 10/2014 v_01 2 non descanned detection (ndd) the use of non-descanned detectors with the lsm 880 nlo is only useful in combination with the nlo laser. The non-descanned detection modules can be us...

  • Page 639

    Chapter 5 - multiphoton laser scanning … lsm 880 non descanned detection (ndd) zeiss 10/2014 v_01 000000-2071-464 13 2.2 mounting and dismounting of ndd modules from the ndd port to take off a ndd module from its port or from the ndd module before loosen the screws (fig. 6/ 1 und 2 ) on the port con...

  • Page 640

    Chapter 5 - multiphoton laser scanning … zeiss non descanned detection (ndd) lsm 880 14 000000-2071-464 10/2014 v_01 the ndd detectors need to be connected to the systems in the following way: lsm 880 nlo lvds a (lsm 880 detection) lvds b t-pmt (via lsm 880 scanner) lvds c reflected light ndds (foll...

  • Page 641

    Chapter 5 - multiphoton laser scanning … lsm 880 non descanned detection (ndd) zeiss 10/2014 v_01 000000-2071-464 15 in addition the power connections have to be made in the following way to the plugs on the back of the electronics rack: fig. 9 power connections of ndd detectors and the scan modules...

  • Page 642

    Chapter 5 - multiphoton laser scanning … zeiss non descanned detection (ndd) lsm 880 16 000000-2071-464 10/2014 v_01 2.4 filter for non descanned detectors (ndds) to make the best use of ndds it is necessary to achieve optimal blocking of the excitation laser light. This is done in the same way for ...

  • Page 643

    Chapter 5 - multiphoton laser scanning … lsm 880 non descanned detection (ndd) zeiss 10/2014 v_01 000000-2071-464 17 fig. 11 example for a filter equipment of a four channel ndd set up on axio observer starting with an ndd module type c or d for lsm nlo systems on axio imager (ndds are only availabl...

  • Page 644

    Chapter 5 - multiphoton laser scanning … zeiss non descanned detection (ndd) lsm 880 18 000000-2071-464 10/2014 v_01 for example, to add a fourth channel to the cascade shown in fig. 13 with a filter combination to additionally detect an emission signal in the far red range the filters have to be re...

  • Page 645

    Chapter 5 - multiphoton laser scanning … lsm 880 non descanned detection (ndd) zeiss 10/2014 v_01 000000-2071-464 19 for axio examiner and axio imager: big.2 with gaasp at reflected light ndd port big ndd as channel 1+2 ndd module type a, 2 channels for upright microscopes, big ndd module as channel...

  • Page 646

    Chapter 5 - multiphoton laser scanning … zeiss non descanned detection (ndd) lsm 880 20 000000-2071-464 10/2014 v_01 big ndd as channel 1+2 ndd module type c, 2 channels for inverted microscopes, big ndd module as channel 3+4 big.2 with gaasp at transmitted light ndd port external detection module b...

  • Page 647

    Chapter 5 - multiphoton laser scanning … lsm 880 non descanned detection (ndd) zeiss 10/2014 v_01 000000-2071-464 21 gaasp ndd 2 channels for axio examiner this detector module is located in a special objective holder. It replaces a standard objective holder on the axio examiner. It holds one object...

  • Page 648

    Chapter 5 - multiphoton laser scanning … zeiss non descanned detection (ndd) lsm 880 22 000000-2071-464 10/2014 v_01 2.5 changing the filter modules big.2 you can remove and replace filter modules by push and click. When opening the filter insert, the safety control will switch off the laser light. ...

  • Page 649

    Carl zeiss confocal laser scanning microscopy c o n fo ca l la se r s ca n n in g m ic ro sc o p y c ar l z ei ss this monograph comprehensively deals with the quality parameters of resolution, depth discrimination, noise and digitization, as well as their mutual interaction. The set of equations pr...

  • Page 651

    Confocal laser scanning microscopy stefan wilhelm carl zeiss microscopy gmbh · carl-zeiss-promenade 10 · 07745 jena · germany.

  • Page 652

    2 page introduction 4 opticalimageformation part 1 deals with the purely optical conditions in a confocal lsm and the influence of the pin - hole on image formation. From this, ideal values for resolution and optical slice thickness are derived. Pointspreadfunction 8 resolutionandconfocality 10 reso...

  • Page 653

    In recent years, the confocal laser scanning microscope (lsm) has become widely established as a research instrument. The present brochure aims at giving a scientifically sound survey of the special nature of image formation in a confocal lsm. 3.

  • Page 654: Introduction

    4 fig. 1 the quality of the image generated in a confocal lsm is not only influenced by the optics (as in a conventional microscope), but also, e.G., by the confocal aperture (pinhole) and by the digitization of the object information (pixel size). Another important factor is noise (laser noise, or ...

  • Page 655

    Scanningprocess in a conventional light microscope, object-to-image trans- formation takes place simultaneously and parallel for all object points. By contrast, the specimen in a confocal lsm is irradiated in a pointwise fashion, i.E. Serially, and the physi- cal inter action between the laser light...

  • Page 656

    6 fig. 3 non-confocal (left) and confocal (right) image of a triple-labeled cell aggregate (mouse intestine section). In the non-confocal image, specimen planes outside the focal plane degrade the information of interest from the focal plane, and differently stained specimen details appear in mixed ...

  • Page 657

    3 rd dimension in addition to the possibility to observe a single plane (or slice) of a thick specimen in good contrast, optical section- ing allows a great number of slices to be cut and recorded at different z-planes of the specimen, with the specimen being moved along the optical axis by controll...

  • Page 658: Point Spread Function

    8 point spread function in order to understand the optical performance charac- teristics of a confocal lsm in detail, it is necessary to have a closer look at the fundamental optical phenomena re- sulting from the geometry of the confocal beam path. As mentioned before, what is most essential about ...

  • Page 659

    The total psf (psf tot ) of a confocal microscope behind the pinhole is composed of the psfs of the illuminating beam path (psf ill ; point illumination) and the detection beam path (psf det ; point detection). Mathematically, this relationship can be described as follows: psf ill corresponds to the...

  • Page 660: Resolution and Confocality

    10 fig. 7 geometric-optical (a) and wave-optical confocality (c) [xz view]. The pinhole diameter decreases from (a) to (c). Accordingly, psfdet (green) shrinks until it approaches the order of magnitude of psfill (red). Resolution and confocality wherever quantitative data on the resolving power and...

  • Page 661

    Resolution resolution, in case of large pinhole diameters (ph >1au), is meant to express the separate visibility, both laterally and axially, of points during the scanning process. Imagine an object consisting of individual points: all points spaced closer than the extension of psf ill are blurred (...

  • Page 662

    12 fig. 8 isophote diagram of the intensity distribution around the illumination-side focus (psfill). The intensity at the focus is normalized as 1. (born & wolf, priniples of optics, 6th edition 1988, pergamon press) let the statements made on the psf so far be further illus- trated by the figure o...

  • Page 663

    Geometricopticalconfocality optical slice thickness (depth discrimination) and stray light suppression (contrast improvement) are basic properties of a confocal lsm, even if the pinhole diameter is not an ideal point (i.E. Not infinitely small). In this case, both depth discrimination and stray ligh...

  • Page 664

    Thus, equations (2) and (3) for the widths of the axial and lateral half-intensity areas are transformed into: 14 1 for rough estimates, the expression λ ≈ √λ em · λ exc suffices. Wave-opticalconfocality if the pinhole is closed down to a diameter of tually “infinitely small”), the character of the ...

  • Page 665

    From equations (7) and (7a) it is evident that depth resolu- tion varies linearly with the refractive index n of the immer- sion liquid and with the square of the inverse value of the numerical aperture of the objective lens {na = n · sin( α )}. To achieve high depth discrimination, it is important,...

  • Page 666

    16 fig. 11 a) variation of pinhole diameter b) variation of numerical aperture d) variation of refractive index c) variation of wavelength ( λ ) fluorescent plane fluorescent point reflecting plane (mirror) optical slice (na = 1.3; n = 1.52; λ = 496 nm) 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 0.5 1.0 ...

  • Page 667: Overview

    17 overview all data in the table refer to quantities in the object space and apply to a fluorescent point object. 1) ph conventional microscopy 1. Optical slice thickness not definable with a conventional microscope, unlike in confo- cal microscopy, sharply defined images of “thick” biological spec...

  • Page 668: Sampling and

    18 sampling and digitization after the optical phenomena have been discussed in part 1, part 2 takes a closer look at how the digitizing process and system-inherent sources of noise limit the performance of the system . As stated in part 1, a confocal lsm scans the specimen surface point by point. T...

  • Page 669

    The object information gained by the scanning process is, in most cases, detected by a photomultiplier tube (pmt), which registers the spatial changes of object properties i(x) as a temporal intensity fluctuation i(t). Spatial and temporal coordinates are related to each other by the speed of the sc...

  • Page 670

    20 if the number of sampling points per feature size is smaller than that given by the nyquist theorem (undersampling), part of the information will be lost. This is evident in figure 14c especially by the unresolved fine features. A greater number of sampling points per feature size (overs- ampling...

  • Page 671

    Pixelsize a quantity of decisive importance in this connection is the maximum scanning angle set via the scanning zoom. By varying the scanning angle, it is possible to directly influence the edge length of the scanned field in the intermediate image plane (or object plane), and thus the pixel size ...

  • Page 672: Noise

    With a pinhole diameter point separation thanks to a deeper dip), which is penalized by a drastic loss in energy. Moreover, it should be considered that it depends on the signal level wich noise source dominates. With high-ampli- tude signals (number of detected photons >10.000), laser noise is the ...

  • Page 673

    Resolutionandshotnoise– resolutionprobability if the number of photons detected (n) is below 1000, fluo- rescence emission should be treated as a stochastic rather than a continuous process; it is necessary, via the shot noise, to take the quantum nature of light into account (the light flux is rega...

  • Page 674

    The pinhole diameter selected in practice will therefore al- ways be a trade-off between two quality parameters: noise (snr as a function of the intensity of the detected light) and resolution (or depth discrimination). The pinhole always needs a certain minimum aperture to allow a minimum of radiat...

  • Page 675

    Possibilitiestoimprovesnr pinhole diameters providing a resolution probability below 90% may still yield useful images if one uses a longer pixel time or employs the signal averaging function. In the for- mer case, additional photons are collected at each pixel; in the latter case, each line of the ...

  • Page 676

    The pictures on the left demonstrate the influence of pixel time and averaging on snr; object details can be made out much better if the pixel time increases or averaging is employed. Another sizeable factor influencing the snr of an image is the efficiency of the detection beam path. This can be di...

  • Page 677: Glossary

    27 glossary a aperture angle of a microscope objective au airy unit (diameter of airy disc) dpix pixel size in the object plane fwhm full width at half maximum of an intensity distribution (e.G. Optical slice) n refractive index of an immersion liquid na numerical aperture of a microscope objective ...

  • Page 679

    To give some further insight into laser scanning microscopy, the following pages treat several aspects of particular importance for practical work with a laser scanning microscope. Pupil illumination optical coordinates fluorescence sources of noise part 3 details.

  • Page 680: Pupil Illumination

    I pupil illumination to a truncation factor t=1.3. The lateral coordinate is nor- malized in airy units (au). From t=3, the airy character is predominating to a degree that a further increase in the truncation factor no longer produces a gain in resolution. Because of the symmetry of the point image...

  • Page 681: Optical Coordinates

    Ii optical coordinates analogously, a sensible way of normalization in the axial direction is in terms of multiples of the wave-optical depth of field. Proceeding from the rayleigh criterion, the follow- ing expression is known as rayleigh unit (ru): the ru is used primarily for a generally valid re...

  • Page 682: Fluorescence

    Iii fluorescence in principle, the number of photons emitted increases with the intensity of excitation. However, the limiting parameter is the maximum emission rate of the fluorochrome mole- cule, i.E. The number of photons emittable per unit of time. The maximum emission rate is determined by the ...

  • Page 683

    Iv with λ = 488 nm and na = 1.2 the sampling volume can be calculated to be v=12.7·10 –18 l. Assuming a dye concentra- tion of 0.01 µmol/l, the sampling volume contains about 80 dye molecules. This corresponds to a number of about 260 photoelectrons/pixel. With the concentration reduced to 1 nmol/l,...

  • Page 684: Sources of Noise

    V sources of noise darknoise dark noise is due to the generation of thermal dark elec- trons n d , irrespective of whether the sensor is irradiated. Nd statistically fluctuates about n d . Dark noise is specified for a pmt voltage of 1000 v; with lower voltages it progres- sively loses significance....

  • Page 685: Literature

    Literature 1.Beyer,h., handbuch der mikroskopie, 2nd edition, veb verlag technik berlin, (1985) 2.Born&wolf, priniples of optics, 6th edition 1988, pergamon press 3.Brismar,h.,trepte,o.,ulfhake,b.: spectra and fluorescence lifetimes of lissamine, rhodamine etc....: influences of some environmental f...

  • Page 686

    In fo rm ati on s ubj ec t to c ha ng e. Pr in te d on en vir on m en ta lly f rie nd ly pa pe r bl ea che d w ithou t c hl or in e. 60 -1 -00 30/ e – pr int ed 1 1. 10 is bn : 9 78 -3 -940885 -02 -9 we gratefully acknowledge the assistance of bernhard gröbler, hartmut heinz and all other staff memb...

  • Page 689

    Carl zeiss confocal laser scanning microscopy this monograph comprehensively deals with the quality parameters of resolution, depth discrimination, noise and digitization, as well as their mutual interaction. The set of equations presented allows in-depth theoretical investigations into the feasibil...

  • Page 690

    Carl zeiss confocal laser scanning microscopy c o n fo ca l la se r s ca n n in g m ic ro sc o p y c ar l z ei ss this monograph comprehensively deals with the quality parameters of resolution, depth discrimination, noise and digitization, as well as their mutual interaction. The set of equations pr...