Internal Medicine Imaging Core Emory University Department of Medicine 1
OPERATION OF THE ZEISS LSM 510 META YOU MUST SIGN UP TO USE THE MICROSCOPE OR COMPUTER EVERY TIME NO EXCEPTIONS Before attempting to use the microscope: you should read information on fixatives for biological material, on photobleaching and anti-fade agents, and on mounting media so that your samples are well-prepared and artifacts are minimized. See Confocal Notebook under the file Preparation of Sample. It is best to grow specimens on glass slides or coverslips. you should also know the excitation and emission spectra for your particular fluorochromes so that you can select the proper configurations available in the software. See Confocal Notebook under the file Selection of Fluorochromes and Selection of Filters. The Zeiss LSM 510 META has three lasers with the following emission lines for excitation: (1) a BLUE DIODE LASER: 405 nm; (2) an ARGON LASER: 458, 477, 488 and 514 nm; (3) a GREEN HeNe LASER: 543 nm; and (4) a RED HeNe LASER: 633 nm. you should familiarize yourself with the choices of objectives on the microscope and the meanings of the markings on the objectives (e.g., the magnification, the numerical aperture (NA), the coverslip thickness requirements). See Confocal Notebook under the file Objectives to learn these markings and their meaning. A Zeiss LSM 510 META OPERATING MANUAL is available on the book shelf to read for more detailed discussions of the microscope operation. Also available are the revised edition of HANDBOOK OF BIOLOGICAL CONFOCAL MICROSCOPY edited by James B. Pawley for reading on the theory of confocal imaging systems (a newer 2 nd edition is available from the Emory Woodruff Library) and CONFOCAL MICROSCOPY FOR BIOLOGISTS by Alan Hibbs. A good introduction to light microscopy is OPTIMIZING LIGHT MICROSCOPY FOR BIOLOGICAL AND CLINICAL LABORATORIES by Barbara Foster available on book shelf (Hilenski personal copy, so please return). A good introduction to image acquisition is North, A.J. 2006. Seeing is believing? A beginners guide to practical pitfalls in image acquisition. J Cell Biol 172:9-18. Copies are also available in the Imaging Core. What users should consider purchasing: Mounting Media Fluoromount G #17984-25 (Electron Microscopy Sciences, P.O. Box 251, 321 Morris Road, Ft. Washington, PA 19034; Phone 215-646-1566; www.emsdiasum.com) Vectashield #H-1000 (Vector Laboratories, Inc., 30 Ingold Road, Burlingame, CA 94010; Phone 650-697-3600 (10 ml=$45.00 as of 8/2001) 2
SlowFade Antifade Kit # S-2828 (Molecular Probes, Eugene, OR) Glass Coverslips (# 1.5) Glass Slides Blank CDs and DVDs (preferably Taiyo Yuden brand) Clear Fingernail Polish (Wet n Wild Clear Nail Protector is recommended other brands contain ethyl acetate/acetone which will lead to loss of fluorescence signal) Glass Bottom 35 mm Dishes (MatTek Corporation, 200 Homer Ave., Ashland, MA 01721; Phone 508-881-6771; FAX 508-879-1532) (used for viewing live cells/living tissue in a fluid) Chambered coverglasses (available from Applied Scientific at 650-244- 9851 or http://www.appliedsci.com for part # s AS-4851 one-chamber; AS- 4852 two- chambers; AS-4854 four-chambers; AS-4858 eight-chambers) (used for viewing live cells) NOTE: Petri dishes are NOT optically clear and can only be used for low magnification (10X, 20X) work. Standard chamber slides will NOT work as they have glass slide bottoms which are too thick to be imaged through. IMPORTANT NOTE: Please read the contents of the folder on Digital Image Manipulation containing information from Nature (Guide for Digital Images), Journal of Cell Biology and the Microscopy Society of America on the acquisition, storage and image processing of digital images taken on the Zeiss LSM 510 META system. We advise you to save your original digital images exactly as they were acquired and to record your instrument settings. BEFORE USING THE SYSTEM, YOU MUST CONTACT: LU HILENSKI (WMB 329, 7-8116, lhilens@emory.edu) OR DEBBY MARTINSON (WMB 303, 7-3712, dmart05@emory.edu) FOR A TRAINING SESSION, YOU WILL BE ASSIGNED A USERNAME AND PASSWORD FOR LOGIN. 3
The following instructions are taken from the Zeiss LSM 510 META Operating Manual and from the Wright Cell Imaging Facility (www.uhnresearch.ca/wcif). STARTING THE HARDWARE: 1. Turn on the EXFO X-Cite lamp power switch. The lamp indicator will illuminate and the display will flash for the warm up period of 90 seconds. NOTE: The unit should not be turned off unless the lamp has been on for a minimum of 5 minutes. 2. Sign in the login sheet with your starting time and the hours shown on the X-Cite lamp. 3. Using the REMOTE CONTROL, switch on System/PC to provide power to the microscope and computer. To switch on the system completely, switch on Components to initialize the LSM software. REMOTE CONTROL SWITCH 4
STARTING THE SOFTWARE 1. Log on to the computer using the log on and password provided to you by the Imaging Core staff. 2. Double click the LSM 510 icon on the desktop to start the LSM software. The LSM 510 Switchboard will appear on the screen.. 3. Click Scan New Images in the LSM 510 Switchboard window. For analysis of existing images, please use the confocal workstation in WMB 306 to keep the confocal microscope free for acquiring images. 4. Click on Start Expert Mode in the LSM 510 Switchboard window. Hardware Initialization window will appear briefly. 5
5. The LSM 510-Expert Mode Main menu appears on the screen. CREATING A DATABASE FOR ACQUIRED IMAGES 1. Click on the File button in the Main menu toolbar. 2. Click on the New button in the File subordinate toolbar. The Create New Database window appears. 3. Select drive D: from pull down menu. Create a new folder with your name. If you have a folder already, create new file if needed. TURNING ON THE LASERS 1. Click on the Acquire button on the Main menu to open the Acquire subordinate toolbar. 2. Click the Laser button to open the Laser Control button. 3. Click the appropriate Laser Unit. Click on Standby button for the argon and 405 nm lasers and the On button for the HeNe s. WAIT 5 MINUTES for the argon laser to become ready. 4. When status is Ready, click On button for the argon and 405 nm lasers. 5. For the argon laser, set Output (%) so the Tube Current is ~6 A (~45% of the output power). 6
SETTING THE MICROSCOPE 1. The VIS, TV and LSM buttons switch the beam path and indicate which beam path has been set. 2. Click the VIS button on the Acquire subordinate toolbar to set the microscope for direct observation via the eyepieces of the binocular tube. 3. Click on the Micro button in the Acquire subordinate toolbar to open the Microscope Control window. 4. Select an objective by clicking on the pop-up menu. The selected objective will automatically move into the beam path. NOTE: Do not use air-objectives after an immersion objective without wiping the immersion liquid (water or oil) from the specimen and objectives. 5. Select a fluorescence filter block by clicking on the Reflector or by clicking on the Microscope Settings buttons. 7
6. The X-Cite lamp can be shuttered on and off by the FL button on the body of the microscope on the right hand-side. 7. Once you have found and focused your specimen, redirect the light path by clicking on the LSM button in the LSM software. The microscope will automatically change the filter-position and shutter the light sources. CONFIGURING THE BEAM PATH AND LASERS 1. Click on the LSM button in the Acquire subordinate toolbar for laser scanning. 2. Click on the Config button on the Acquire subordinate toolbar. 3. Click on either Single or Multi Track. Use Single for single, double and triple labeling, simultaneous scanning only. Advantage: faster image acquisition. Disadvantage: cross talk between channels. Use Multi Track for double and triple labeling, sequential scanning, line by line or frame or frame. Advantage: when one track is active, only one detector and one laser are switched on, reducing cross-talk. Disadvantage: slower image acquisition. 4. Click Config button in the Configuration Control window to use one of the pre-programmed track configurations. 8
5. Load the configuration from the Channel Mode Configurations that matches your fluorophores (referred to as tracks ). Click on the dropdown box and select a desired configuration, click Apply, then Close. IF YOU CANNOT SEE THE APPROPRIATE CONFIGURATION FOR YOUR FLUOROPHORES, CONTACT THE IMAGING CORE STAFF. SCANNING AN IMAGE 1. Click on the Scan button in the Acquire subordinate toolbar to open the Scan Control window. 2. Set the parameters for scanning by selecting Mode. 9
3. Select Frame, Speed (scan speed 8 usually produces good results), dynamic range of 8 or 12 bit in the Pixel Depth, Scan Direction & Scan Average. NOTE: Publication quality images should be acquired using 12 bit data depth (4096 gray levels). 12 bit is also recommended when doing quantitative measurements or when imaging low fluorescence intensities. HOWEVER, 12 bit files are LARGE. 4. Set the scan averaging to increase the signal/noise ratio. IMAGE ACQUISITION 1. Select Channels in the Scan Control window. For each channel, click on the colored channel button, and then click 1 (for 1 Airy Unit). 2. For each channel, click on the colored channel button, and then click 1. This sets the Pinhole size to 1 (Airy unit) for best results. Pinhole adjustment changes the Optical Slice. NOTE: When collecting multi channel images, adjust the pinholes so that each channel has the same Optical Slice. Increase the pinholes of the shorter wavelength channels to match the longest wavelength. Do not go below 1 Airy unit. This is important for colocalization studies. 3. Start scanning using one of the Find, Fast XY, Single or Cont buttons. 4. Click Stop button to stop the current scan. IMAGE OPTIMIZATION 1. If using Multi Track, it is easier to set the imaging parameters for each Track individually. Switch off each track with the checkbox along side it. Highlight the chosen track. 10
2. Optimizing the confocal settings is best done with the image pseudocolored to highlight saturated and zero value pixels. Click Palette on the Image window tool bar and the Color Palette window will open. 3. Click Range Indicator and close the window. If the scanned image is too bright, it appears red=saturation (maximum). If the image is not bright enough, it appears blue=zero (minimum). 4. While acquiring an image with Fast Scan, adjust Ampl Offset (setting the min signal) so that only a few of the pixels in the background are blue. 5. Set Detector Gain and Excitation power (setting the max signal). Low laser power causes less bleaching but requires the detector gain to be set high, which introduces noise. You may have to adjust the Ampl Offset until all blue pixels disappear, and then make it slightly positive. 6. Reduce the Detector Gain until the red pixels only just disappear. 7. Leave Ampl Gain as 1 unless you have a very dim signal and nothing else works. This will amplify noise as well as signal. REMEMBER: A few red speckles; a few blue speckles in each channel. 8. After you have optimized the first channel, stop scanning by clicking Stop. 9. Turn off the optimized channel in the Configuration Control window. Turn on the next channel and optimize these settings for this channel. 10. Once you are satisfied that each channel detector is set optimally, ensure that all Tracks are checked. 11. Go to Scan Control window and click the Mode button. 11
12. Change the Frame size by clicking on the Optimal button. Noise can be reduced by averaging a number of frames and slowing the scan speed. Try the following: Mode: Line Method: Mean Number: 4 Scan Speed: 8 13. Change the image Palette back to No Palette. 14. Click Start. 15. Save your final image to a database. Click Save as and save image to an existing database (Open MDB button) or create a new database (New MDB button). Do not select Compress file option. 12
SCANNING A Z STACK 1. Select Z Stack in the Scan Control window. 2. Using Fast scan, focus up and down with the microscope focus wheel to ensure your specimen is in the center of the field of view and the microscope is focused in the middle of the specimen. 3. Click Stop acquisition. 4. Click Mark First/Last on the Z Settings panel. Click on the XY cont button. 5. Use the focusing drive of the microscope to focus on the upper position of the specimen area where the Z stack is to start. 6. Click on the Mark First button to set the upper position of the Z stack. 7. Focus on the lower specimen area where the recording of the Z stack is to end. 13
8. Click on the Mark Last button to set this lower position. 9. Click on the X:Y:Z button to set the Z interval in such a way that the voxel has identical dimensions in the X-, Y-, and Z- directions (cube). 10. Click on Z Slice in the Scan Control window to bring up the Optical Slice dialog. 11. Click the Optimal Interval button. Check that the optical section for each channel is the same. If your optical sections are different for each wavelength, go to Mode/Channels and adjust the Pinhole so that each channel has the same optical section around 1 Airy Unit, but one channel will have to be slightly larger. 12. Click on the Start button to start the recording of the Z stack. You can check progress by selecting Gallery button on the image window sidebar. 14
Z ATTENUATION COMPENSATION 1. In the Scan Control: Z Settings window, click on Auto Z Corr. 2. While in Fast XY scan mode, focus to near the top of the sample where it is brightest. You should have already set the detector and laser settings for this slice. If not, go to the Scan Control: Channels window and set the detector gains and laser intensity to get adequate signal at this focus position. 3. In the Scan Control: Z Settings, click Set A. 4. While in the Fast XY scan, manually focus to near the bottom of the Z series. 5. Go to the Scan Control: Channels window and set the detector gains and laser intensity to get adequate signal. 6. Click Set B button in the Scan Control: Z Settings window. 7. Click Start to begin the Z series acquisition. 15
SWITCHING OFF THE SYSTEM 1. Click on File button in the Main menu and then click on the Exit button to exit the LSM software program. 2. To turn off the lasers, click Acquire, then click Laser button in the subtoolbar. 3. In the Laser Control window, click Off for each laser (or to Standby if someone else is using the system within the next two hours). 4. Remove specimen and clean microscope. Wipe off oil from objective using the LENS PAPER provided. 5. Move to a low power objective (10X or 20X). 6. Cover the microscope with the blue Zeiss cover avoiding the hot lamp housing. 7. Tidy up the workspace! 8. Burn your data to CD or DVD. 9. If you are the last one to use the system for the day, wait until the argon laser fan has switched off. 10. Switch off the Components and System/PC on the Remote Control. 11. Switch off the EXFO X-Cite lamp. Record the end time and the lamp hours on the sign-up sheet. OPENING YOUR IMAGES OFFLINE 1. You may use the free Zeiss Image Browser available from: http://www.zeiss.de/lsm Follow link in lower right hand corner: Free LSM Image Browser 2. You may use the full version of the software LSM Image Examiner on the computer in WMB 306. 16