Basic Users Guide for the LSM 510 Confocal Microscope

Transcription

1 Basic Users Guide for the LSM 510 Confocal Microscope Ian Jones and Adam Westmacott Department of Biology & Biochemistry, University of Bath, Bath, UK Updated 01 October 2003 This guide describes the basic operation of the confocal microscope for routine immunocyto- and immunohisto-chemistry (single and multiple fluorescent labelling). It is not intended to be definitive users manual and certainly does not cover all techniques, such as line scans and DIC imaging. Further information and advice can be obtained from the Zeiss manual in the confocal room or from experienced confocal users within the department. The confocal has 3 single photon lasers (Ar, HeNe 543 and HeNe 633) which permit the user to select excitation wavelengths of 458, 488, 514, 543, 568 and 633nm. The LSM software provides a wide range of image processing functions, including standard 2D / 3D (stereo, projection) functions, processing of voxels and 3D measurement functions and parameters.

2 Quick Start Guide Action Page 1. Start up the system (if not already on) 3 2. Switch on required lasers 3 3. Set track laser configurations 4 4. Set microscope configuration 5 5. Orientate and focus sample using microscope (with transmitted or fluorescence illumination) 6 6. Set confocal scan configurations and scan image 6 7. Creation of Z-stacks 8 8. Creating a database 9 9. Putting system into standby mode or shutting down 10 2

3 1. Start Up if the system is switched off (if left on, go to 1.h. or 2.a.) a. Turn system on using the control switch (large black button to the left of the microscope marked Remote Control ). This should switch on the microscope and laser control unit. Turn on the PC using button on top right of tower case (on floor under monitors). b. Switch on the air conditioning unit using the infra-red control (the air conditioning must be on if the lasers are in use to avoid overheating). c. Switch on the fluorescent lamp unit (box to the left of the microscope marked 2788 ). d. Check that the transmitted light unit is on (box to the left of the microscope marked SNT 12V 100W) and the dial is turned up to around 10. e. When prompted press Ctrl+Alt+Del to log on to the computer. f. Press Return at the username / password prompt (do not enter any values). g. Double click on the LSM 510 icon to start confocal operating software. The LSM 510 operating mode panel will appear: h. For scanning, click on Scan New Images and Start Expert Mode (the LSM programme will initialise and open the main menu bar). For analysis of existing images, please use the confocal workstation in 0.44 to keep the confocal microscope free for scanning only (will also save money!). 2. Switch on required lasers a. Click on the Acquire button on the main menu bar and then the Laser button in the subfolder: 3

4 This will open the laser control window: There are three lasers currently connected to the confocal microscope: i. Argon Laser = 488nm (used for FITC, CY2, AlexaFluor488, etc) ii. HeNe1 Laser = 543nm (used for rhodamine, Texas Red, CY3, AlexaFluor543, etc) iii. HeNe2 Laser = 633nm (used for CY5, etc) b. To switch on the Argon laser, highlight the laser name in the control window and click the Standby button. The laser will start to warm up and after one minute a Ready message will appear. Now click on the On button and scroll the laser intensity to 75%. The HeNe lasers can be switched on directly by highlighting them individually in the control window and clicking the On button(s). 3. Set track laser configurations a. Choose the required laser path settings (e.g. single FITC or double FITC + Rhodamine) using one of the pre-programmed track configurations: Click on the Config button in the Acquire subfolder on the main menu bar. The configuration control window will appear: 4

5 For single fluorochrome visualisation, click on Single Track, and for multiple fluorochrome visualisation, click on Multi Track. In either case, click on the Config icon on right side of window and choose the appropriate track configuration from the resulting drop-down and finally click Apply : 4. Set microscope configuration a. To set up the microscope, click on Acquire on the main menu bar and then the Micro button in the subfolder. This will open the Axiovert control window: b. Choose the appropriate objective lens by clicking on objective icon and selecting from the resulting drop-down menu (note, there are two 63X lenses, one for oil-immersion and one water-immersion choose the correct one!). It is good practise to lower the objective lens to its bottom position using the focussing wheels on the side of the microscope prior to installing a new sample. To save time, there is a course adjustment button on the left of the microscope by the focussing wheel (small black button). This is an on-off button so must be pressed again to return to fine focussing. 5

6 c. If necessary, place a small drop of oil on the objective lens (make sure that it is an oilimmersion lens first!) then place the slide to be analysed upside-down on the stage and secure in place using the sliding clamps. Align the sample on the slide over the objective lens using stage control wheels on the left of the microscope. 5. Orientate and focus sample using microscope (with transmitted or fluorescence illumination) a. To view specimens directly down the microscope itself, pull out the lower of the two sliders on the right hand face of the microscope (there is a diagram by the sliders indicating slider position for different viewing modes). b. For transmitted light, click the Transmitted light button at the top of the Axiovert control window (see previous page). Switch light on and use the neighbouring scroll bar to vary the light intensity accordingly. Use the focus wheels to raise the objective lens until the specimen is in focus. c. For fluorescence imaging, switch off the transmitted light by clicking the button in the Axiovert control window (if required) and choose the appropriate filter from the Reflector drop down menu (in the same window): i. Fset09 = 488nm (used for FITC, CY2, AlexaFluor488, etc) ii. Fset15 = 543nm (used for used for rhodamine, Texas Red, CY3, AlexaFluor543, etc). (note, fluorochromes excited by the HeNe2 laser (e.g. CY5) cannot be viewed directly down the microscope). 6. Set confocal scan configurations and scan image a. Push in the bottom slider on the right side of the microscope (if not already in) to prepare the microscope for scanning mode. b. To scan an image, click on the Scan button in the Acquire subfolder on the main menu bar. The scan control window will appear: 6

7 For a fast scan, click Fast XY on the right hand side of the scan control window (an image window will appear). To view the individual channels of a multi-track image, click Split XY in the image window (return to a single image by clicking XY ). Ensure that your sample is in focus using the focus wheel and work fast as the fluorescence may bleach with time. c. To optimise the image, click on Channels in the scan control window and click on the relevant channel that you wish to alter (Ch1, Ch2 or Ch3), then click Find on the right hand side of the window. The software will automatically determine the optimal settings for your sample. d. If necessary, further optimise the image by adjusting the scroll bars in the scan control window ( Channels sub-folder): i. Detector gain sets brightness/contrast ii. Ampl. offset adjusts background iii. Ampl. gain alters amplification factor e. Normally there is no need to change the excitation of the track or the pinhole setting as these have been optimised for standard samples. On occasion, however, this may be necessary (e.g. for weakly fluorescent samples) and these parameters can be altered using the scroll bars in the scan control window ( Channels sub-folder). f. For multi-tracking, repeat steps d - f for each of the remaining channels in use. g. To further improve image quality, open the Mode sub-folder in the scan control window (see page 6) and then click on the Frame button. Choose an appropriate frame size (512x512 pixels for quick scan, 1024x1024 pixels for high-quality scan), a low scan speed (5 or 6) and 7

8 the number of averages (1 for quick scan, 4 or 8 for high-quality scan). Magnify, move and rotate the image if required using the buttons at the bottom of the window. The other functions in this window usually do not require altering for a standard frame scan. h. When the image has been optimised, click Stop in the scan control window and then on Single to generate a single image. To store this image in your database, click on the Save As button on the right of the image window (see section 8 for guide to creating a database). The image can be annotated (size bar, etc) using the workstation in 0.44 (or you can download a free basic image manipulation programme from the Zeiss web site and do this on your own PC). i. Images can also be exported as graphics files (JPEG, TIF, etc). For this, select the image to be exported and click Export on the main menu bar. 7. Creation of Z-stacks a. Click on the Z-Stack button in the scan control window (this will open the Z settings panel) and then click on Fast XY. Use the focus wheel to focus on the top of the sample, where the Z-stack is to start, and click on Mark First in the Z settings panel. Now focus on the bottom of the image, where the Z-stack is to end, and click on Mark Last. b. Click on Z-Slice in the Z settings panel to set the optimal interval and pinhole diameter(s) for the stack. If necessary, change the image pixel size and number of averages in the Mode sub-folder, then click on Start to create the stack. 8

9 c. To create a 3D projection of the Z-stack, open the 3D View folder on the main menu bar and click on Projection. In the projection window, select the image to be projected, the first angle, number of projections and difference angle, then click Apply. The projection will appear in a new image window and can be animated by clicking on the appropriate button in the image window. 8. Creating a database a. To create a new database for saving scanned images to, click on File on the main menu bar and then the New button in the subfolder. Databases should preferably be created either on a removable disk (e.g. MO disk) or on an external computer (connected to via network neighbourhood) and not the confocal hard disks. Enter a database name, destination folder and click Create. 9

10 b. To open an existing database, click on File on the main menu bar and then the Open button in the subfolder. 9. Putting system into standby mode or shutting system down a. Make sure that any oil is removed from the oil-immersion objectives using the lens tissue provided. Tidy the workspace around the microscope and fill out the log sheets with your name, confocal usage time, lab number, grant code and any comments. b. If the confocal is booked after you (or within one hour), close all windows except the Main menu bar and Laser subfolder and place the Argon laser in Standby Mode. Do not switch the Argon laser off otherwise then next user must wait 35 minutes before it can be switched back on again. If no-one is booked to use the confocal for at least an hour, switch off the argon laser and the fluorescent and transmitted lamp units (see 1.b. and 1.c.). c. If you are the last user for the day, switch off the lasers, close all windows, turn off the fluorescent and transmitted lamp units and shutdown the computer. After 5 minutes (to allow the lasers to cool down), the system can be switched off using the main remote switch (see 1.a.). Finally, cover the microscope, switch off the air conditioning and lock the room as you leave. 10