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Microscopy from Carl Zeiss
Quick Guide
LSM 710 / LSM 710 NLO
and ConfoCor 3
Laser Scanning Microscopes
LSM Software ZEN 2008
August 2008
We make it visible.
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Contents Page Contents ................................................................................................................................. 1 Introduction............................................................................................................................ 2 Starting the System ............................................................................................................... 3 Introduction to ZEN – Efficient Navigation ....................
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Introduction This LSM 710 / LSM 710 NLO and ConfoCor 3 Quick Guide describes the basic operation of the LSM 710 / LSM 710 NLO and ConfoCor 3 Laser Scanning microscopes with the ZEN 2008 software. The purpose of this document is to guide the user to get started with the system as quick as possible in order to obtain some first images from his samples. This Quick Guide does NOT replace the detailed information available in the full user manual or in the manual of the respective mic
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0 laser run Laser - Fertigung GmbH Starting the System Switching on the LSM system • Switch on the main switch (Fig. 1/1) and the safety lock (Fig. 1/2). • When set to ON the power remote switch labeled System/PC provides power to the computer. This allows use of the computer and ZEN software offline • To completely switch on the system, now press the Components switch to ON. This starts the other components and the complete system is ready to be initialized by the ZEN software.
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Starting the ZEN software • Double click the ZEN 2008 icon on the WINDOWS desktop to start the Carl Zeiss LSM software. The ZEN Main Application Window and the LSM 710 Startup window appear on the screen (Fig. 3) Fig. 3 ZEN Main Application window at Startup (a) and the LSM 710 Startup window (b and c) In the small startup window, choose either to start the system online ("Start System" hardware for acquiring new images) or in Image Processing mode to edit already existing ima
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Fig. 4 ZEN Main Application Window after Startup with empty image container Fig. 5 ZEN Main Application Window after Startup with several images loaded 05/2008 5
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Introduction to ZEN – Efficient Navigation ZEN - Efficient Navigation - is the new software for the LSM Systems from Carl Zeiss. With the launch of this software in 2007 Carl Zeiss sets new standards in application-friendly software for Laser Scanning Microscopy. The ZEN 2008 Interface is clearly structured and follows the typical workflow of the experiments performed with confocal microscopy systems: On the Left Tool Area (Fig. 4/D) the user finds the tools for image acquisition, im
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The Pro-Basic concept ensures that tool panels are never more complex than needed. In Basic Mode, the most commonly used tools are displayed. For each tool, the user can activate Pro Mode to display and use additional functionality (Fig. 6). Fig. 7 ZEN Window Layout configuration More features of ZEN 2008 include: • The user can add more columns to the Left Tool Area or detach individual tools to position them anywhere on the monitor. To add a column, drag a tool group by the title b
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Setting up a new image document and saving your data To create a new image document in an empty image container, click the "Single" or the "Start" button. The new document is immediately presented in the Open Images Area. Remember, an unsaved 2D image in the active image tab will be over-written by a new scan. Multi-dimensional scans or saved images will never be over-written and a new scan will then automatically create a new image document. Alternatively you can create a new empt
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Advanced data browsing is available through the File Browser (Ctrl-F or from the File Menu). The File Browser can be used like the WINDOWS program file browser. Images can be opened by a double-click and image acquisition parameters are displayed with the thumbnails (Fig. 9). For more information on data browsing please refer to the detailed operating manual. Fig. 9 File Browser 05/2008 9
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Turning on the lasers • Open the Laser Tool in the left tool area (always the first in the list) and activate the lasers you need for your experiment (Fig. 10). Remember, the argon multi-line laser has to first be put to standby for a 5 minute warm-up before it changes to on. • Zeiss recommends operating the Argon multi-line Laser at a tube current of about 6 A ( ∼50 % output). This is the best compromise between laser stability/power and laser life-time. (tube-current control can be
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Setting up the microscope Changing between direct observation, camera detection and laser scanning mode The Ocular, Camera and LSM buttons switch the beam path and indicate which beam path is currently in use for the microscope: • Click on the Ocular button to change the microscope beam path for direct observation via the eyepieces of the binocular tube, lasers are blocked. • Click on the Camera button to change the beam path of the microscope for the port where the camera is att
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Setting the microscope for reflected light • Click on the Reflected Light icon to open the X-Cite 120 Controls and turn it on. • Click on the Reflected Light shutter to open the shutter of the X-Cite 120 lamp / HBO100. • Click on the Reflector button and select the desired filter set by clicking on it. Storing the microscope settings Microscope settings can be stored as configurations (Fig. 13) by typing a config name in the pull-down selector and pressing the save button. Fa
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Configuring the beam path and lasers • Click on the LSM button. Choosing a configuration Simultaneous scanning of single, double and triple labeling: − Advantage: faster image acquisition − Disadvantage: Eventual cross-talk between channels Sequential scanning of double and triple labeling; line-by-line or frame-by-frame: − Advantage: Only one detector and one laser are switched on at any one time. This reduces cross- talk. − Disadvantage: slower image acquisition • Open the I
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Settings for track configuration in Channel Mode • Select Channel Mode if necessary (Fig. 15). • Click on the LSM tab (Fig. 14). The Light Path tool displays the selected track configuration which is used for the scan procedure. • You can change the settings of this panel using the following function elements: Fig. 15 Imaging Setup tool for a single track (LSM) Activation / deactivation of the excitation wavelengths (check box) and setting of excitation intensities
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• For storing a new configuration enter a desired name in the first line of the Configurations list box (Fig. 17) and click Store. • For loading an existing configuration select it from the list box and click on the Load button. • For deleting an existing configuration select it in Fig. 17 Track Configurations window the list box and click on Delete. Settings for multiple track configurations in Channel Mode Multiple track set-ups for sequential scanning can be defined a
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Scanning an image Setting the parameters for scanning • Select the Acquisition Mode tool from the Left Tool Area (Fig. 18). • Select the Frame Size as predefined number of pixels or enter your own values (e.g. 300 x 600) in the Acquisition Mode tool. Click on the Optimal button for calculation of appropriate number of pixels depending on objective N.A. and λ. The number of pixels influences the image resolution! → Fig. 18 Acquisition Mode tool Note: When using an Axioskop
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Setting scan averaging Averaging improves the image by increasing the signal-to-noise ratio. Averaging scans can be carried out line-by-line or frame-by-frame. Frame averaging helps to reduce photo-bleaching, but does not give quite as smooth of an image. • For averaging, select the Line or Frame mode in the Acquisition Mode tool. • Select the number of lines or frames to average. Adjusting pinhole size • Select the Channels tool in the Left Tool Area. • Set the Pinhole size to
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Image acquisition Once you have set up your parameter as defined in the above section, you can acquire a frame image of your specimen. • Use one of the Find, Fast, Continuous, or Single buttons to start the scanning procedure to acquire an image. • Scanned images are shown in separate windows. • Click on the Stop button to stop the current scan procedure if necessary. Select Find for automatic pre- adjustment of detector gain and offset. Select Fast for continuous fas
