ZEISS LSM510META confocal manual

Transcription

1 ZEISS LSM510META confocal manual Switching on the system 1) Switch on the Remote Control button located on the table to the right of the microscope. This is the main switch for the whole system including the computer. 2) Turn on the computer using the power button on the tower. 3) Once the computer has booted, enter your user name and password. Note: every few months, the system will ask for a renewal of the password. Starting the software 1) Double-click the LSM510 META icon on the desktop 2) the LSM switchboard appears... 3) Make sure Scan New Images is selected. ("Use Existing Images" is an off-line emulation mode). 4) Click on Start Expert in the Switchboard 5) After the software is initialized, switch on the microscope mercury lamp.

2 Main menu: The main menu window looks like this: 1) In the FILE menu, you can create a new Zeiss database for storing the images and the corresponding information about objectives, excitation and emission settings, etc (File>New). The data base has to be stored in your personal folder on D:/users. 2) Selecting the ACQUIRE menu will show the main image acquisition boxes: Laser, Micro, Config, and Scan. Laser control menu: Selecting the Laser button will open the Laser Control with a list of available lasers (Aquire>Laser). Turn on the appropriate laser(s) to excite the fluorophores in your sample: LASER λ FLUOROPHORE Laser Diode nm DAPI/Hoechst Argon 458nm CFP 488nm Alexa488, CY2, FITC, GFP 514nm YFP HeNe1 543nm Alexa546/594, CY3, TRITC, RFP HeNe2 633nm Alexa647, CY5

3 To turn on the Argon laser, click to warm it up. After the initial warm-up period, the on button will appear allowing the laser to be fully activated. Set the output intensity to an appropriate level - usually between 25-40%. The laser should never exceed 50%!! NOTE: The Argon laser requires a special shutdown procedure (see the Shutdown Procedure paragraph). To turn on the Laser Diode 405, or either of the HeNe lasers, simply click the appropriate "On" buttons - no warm-up is required for ignition. We try to minimize the number of times a laser is turned on or off - if somebody will use the laser within 1 hour leave it on (Argon and UV lasers in standby). Check the online booking site to see microscope usage scheduled before and after your session. Turn off any lasers you don't need that may have been left on for you by the previous user. Microscope control menu: Open the microscope control window by clicking Micro in the main software menu (Aquire>Micro). The objectives, fluorescent filters are motorized and can be controlled in the software.

4 First select the objective that is required for the experiment by clicking the objective button. The microscope is equipped with different objectives. The 10x and 20x objectives are air objectives, and should not be spoiled with immersion oil. The 40x and 63x objectives are oil objectives. When one of the oil objectives is selected, use small droplets of immersion oil. Upon changing sample slides, wipe the oil from the objective with special lens paper prior to mounting a new slide. Note: the system is an inverted microscope, so the cover slip on the object slide should face down. For live cell imaging, a small droplet of oil should be put on the objective before inserting the live cell imaging chamber. To view your sample by eye, first select VIS in the main control menu: Select the correct fluor cube by clicking Reflector to look at the fluorescence; FSet02wf Blue, CY3 - Red, FITC special -Green. Click Reflected light for fluorescence. For imaging with the software, close the Reflected light and click

5 Filter Configuration menu: Select the Configuration control menu (Aquire>Config). In the Channel Mode, select Single Track for single channel imaging or simultaneous excitation of multiple channels; and Multi Track for laser scanning in multiple channels sequentially, which can be useful when bleed through is a problem. If a filter configuration is already created, click on the disk symbol Config on the right side of the window to open a list with the track configurations. Click Apply to load the laser & filter settings for that configuration. If you prefer to use a new configuration, there is the possibility to change filters, mirrors, dichroics etc. The new setting can be saved by clicking the disk symbol Config and subsequently Save under a new name. See the paragraph Customizing the Filter Configuration below.

6 Image scanning control Acquire>Scan opens the "Scan Control" window - the main dialog box for acquiring images. There are several adjustments to make to get a good image. In the Mode tab of the Scan control, most of the settings will be fine as the default values. Averaging is the most important thing to adjust to reduce noise. The amount of averaging necessary to obtain a good image depends on the signal to noise ratio of your sample image. Low contrast images will require more averaging but be aware that this could cause photobleaching. Line scan averaging of 4 is sufficient for most samples. Digital zoom (Image cropping) is optional.

7 In the Channels tab, many adjustments can be made. Those adjustments can be loaded, or saved as a configuration setting in the configuration control window ( Config button). Another option is to re-use the settings from previous images ) Channels indicate which configuration is selected in the configuration window. For optimizing the settings, its convenient to select only one track. For obtaining a multitrack image, all tracks have to be selected in the configuration window, and these will be shown here in the scan window as well. 2) The different laser lines are shown here. It is possible to adjust the percentage transmission. For suggested settings, see the table in the Customizing the Filter Configuration paragraph below. 3) The pinhole is the principle for confocality. Opening up the pinhole will result in more light, but it reduces the confocality and resolution. An optimal pinhole for the selected wavelength is Airy Unit = 1, which correspond approximately with a Optical slice of 1 um with the 63x objective. Note: with multi-track imaging, its important to have the same

8 optical slice rather than Airy Unit number. An approach is to set for the longest wavelength the Airy Unit one, which will correspond to a specific optical slice thickness. Adjust the pinhole for the other settings so that those have the same optical slice thickness. 4) With Detector Gain and Amplifier Offset the image intensity and background levels gain be adjusted. Avoid overexposure and underexposure of the image (see the corresponding paragraph below). 5) Clicking Fast XY will open an extra window with fast ( live ) scanning of your image, and its useful to optimize the correct focus and exposures (detector gain, amplifier offset). 6) Clicking Single will result in scanning the final averaged image. For multi track imaging, make sure that all the required tracks are selected in the configuration window. The image window The final averaged image of your specimen is depicted in the image window:

9 The most important thing is to SAVE the image in your Zeiss database which has been created at the start of the session (see above: Main Menu>File>new). Other options are: Chan: showing or omitting the fluorescence channels in the image Zoom: digital zoom Slice: stack slider when the image has different z-planes Overlay: several draw functions including a scale bar Palette: to display the LUT and range indicator (used for determining optimal detection with Detector Gain and Amplifier Offset) Reload: re-use configuration/acquisition settings Crop: zoom in the selection Copy: copying the actual image window to other applications (e.g. powerpoint). XY: Show merge image Split XY: Show channels separately

10 Customizing the Filter Configuration: Perform this only when no available configuration is in the list. The configuration window depicts the light path from laser to specimen, and to the detectors (Ch2, Ch3). The easiest approach to make a new configuration is to follow the light path (from 1 to 5). 5a 3 4 5b 2 1 For every configuration track, follow the following steps. For multi tracks, one needs to add tracks for different fluorophores. For every configuration, the track mode FRAME is recommended. 1) Selecting the appropriate laser by clicking the Excitation button and determine the transmission %.

11 Laser line Dyes Suggested Transmission % * 405 nm Hoechst, DAPI 5-20 % 458 nm CFP 20 % 488 nm Alexa488, CY2, FITC, GFP 1-10% 514 nm YFP 10 % 543 nm Alexa546/594, CY3, TRITC, 20 % RFP 633 nm Alexa647, CY % * Those values are suggestions, but can be adjusted by the user 2) The dichroics have to be selected according to the excitation laser line. HFT dichroics reflect the indicated wavelengths, all other wavelengths pass. For example, select HFT 405/488/543 for Hoechst, GFP, RFP multi track imaging, or HFT488 for GFP single track imaging. 3) The mirror directs the emission light towards the detectors Ch2/Ch3. When the META system (ChS) is being used, then the mirror should not be selected. 4) Depending on the fluorophore, the emission light should either be directed to Ch2 or 3 (different filter sets). NFT reflects the indicated and shorter wavelengths, while higher wavelengths pass.

12 5) Select the appropriate filter set for detection of the emitted light: Filters for Ch2 Filters for Ch3 Recommendation: FLUOROPHORE DETECTOR FILTER Hoechst/DAPI Ch2 BP CFP Ch2 BP Alexa488, CY2, FITC, GFP Ch3 BP YFP Ch3 BP Alexa546/594, CY3, TRITC, Ch3 BP RFP Alexa647, CY5 Ch3 LP 650 BP: wavelengths of the indicated range pass, others are blocked LP: the indicated and longer wavelengths pass, shorter wavelengths are blocked

13 Bleach Control: The Bleach control window only needs to be opened when performing fluorescence bleaching, DNA damage, and photoactivation experiments. Premade settings can be selected from the list and applied. For new settings (which can be stored) several basic options are available. Bleach after number scans: This allows in time lapse imaging to start bleaching after the indicated Scan number, so imaging pre-bleach settings. This option works only in combination with the Time Control window when StartB or MeanROI is clicked (see below). Bleach repeat after number scans: This defines how frequent the bleach pulse is applied. This option works only in combination with the Time Control window when StartB or MeanROI is clicked (see below). Iteration: Defines the number of bleaching scans in the selected region. Define Region: Opens the EditROI enabling to create a region of interest in the image window. The bleaching will occur only in this region. Laser Lines: defining which laser lines and intensities should be used.

14 Edit ROI: The Edit ROI window is mainly used in combination with Bleach Control and Time Series Control. Here any type of region can be selected and drawn in the image. This region will be used for laser bleach pulses (Bleach Control) and for recording, which can be selected in the Scan Control window as Use ROI.

15 Time Series Control: This window is only used when time lapse imaging experiments are performed. Several options are available, but the main ones are: Stop Series Number: defines the number of image acquisitions Cycle Delay Time, Unit: defines the time between imaging StartT : Start the time lapse imaging StartB: Start the time lapse imaging in combination with the Bleach Control MeanROI: Measuring the fluorescence intensity in the ROI over time. No images are taken.

16 Shutdown Procedure Before shutting down the whole system after your session, check the online booking system ( to check if there is another user scheduled after your session. If there is a session within 1 hour, then close all windows and log off from Windows. Clean the working area and the oil objectives in case you have used them. If the following session is more than 1 hour later, perform the shutdown procedure: - In the laser control menu, switch all lasers off. The Argon laser should be cooled for approx more minutes. So wait with proceeding until the cooling unit stopped (the fan noise is a good indicator). - Close the software, and shutdown Windows. - Switch off the mercury lamp - Switch the whole system off with the remote control. And finally, fill in the user sheet.