Operating Instructions for Zeiss LSM 510 Location: GNL 6.312q (BSL3) Questions? Contact: Maxim Ivannikov, maivanni@utmb.edu 1
Attend A Complementary Training Before Using The Microscope All future users should be first trained on an identical microscope located in BSB 4.328 before using this confocal microscope. The training is complementary to BSL3 users. To set up your training and for any other questions, please contact: Maxim Ivannikov, maivanni@utmb.edu. 2
1. Powering up the microscope 1.1) Turn on the mercury lamp 1.2) Power the PC 1.3) Turn on the remote SWITCH #1 SWITCH #2 SWITCH #3 3
2. Starting Acquisition Image Manager (AIM) 2.1) Windows loads without a password. On the desktop click on the LSM510 icon. 2.2) To load the acquisition mode of AIM, click first on Scan New Images and then on Start Expert Mode. LSM 510 2.3) The expert mode menu strip is loaded. Click on the Acquire button. 4
3. Switching on the lasers to be used 3.1) Click on the Laser button to open the Laser Control window. 3.2) In the Laser Control window select laser(s) based on the excitation wavelengths of fluorophores you will be imaging. Power the lasers on by clicking on either Standby or On button. If the Argon laser is used, bring the output (%) slider to 50%. The 405 laser will power on automatically. Click Close. Tip: DAPI needs 405 nm (violet) laser Alexa 488 & EGFP need 488 nm (blue) laser Alexa 555-594 & tdtomato need 543 nm (green) laser Alexa 633-647 & Cy5 need 633 nm (red) laser 5
4. Choosing an objective 4.1) Click on the VIS button to enable viewing of your sample with oculars/eyepieces. Then, click on the Micro button to open the Microscope Control window. 4.2) In the open window, click on the objective icon to open a drop-down objective selection menu. Select an objective based on desired detail resolution (objective s NA) and optical magnification (MAGx). Tip: The microscope has 5 objectives, 2 water (10x, 63x) and 1 oil immersion (100x) and 2 air (20x, 40x) objectives. Example: C-Apochromat 63x/1.2W corr: 63x magnification, 1.2 NA, Water immersion objective with a corrective collar (used to adjust to your coverslip thickness). 6
5. Focusing on your sample 5.1) If using an immersion objective, apply a drop of either filtered water OR oil to the front lens of an immersion objective. Do not apply anything onto air objectives! 5.2) Click on the Stage icon to bring up the Stage and Focus Control window. 7 5.3) In the Stage and Focus Control window, click on the load button to lower the objective all the way down. Click the Close button. 5.4) Place your sample onto the stage with coverslip down. Bring the objective slowly up using coarse focus knob on the microscope body until the coverslip just touches the immersion media. Stop. Important: If the microscope makes a beeping sound when you try to raise the objective. Keep pressing on the button on the right side of the microscope while moving the focus knob to override the focus lock.
5. Focusing on your sample (cont.) 5.5) Navigate to the Microscope Control window (see step 4.1). Based on fluorophores in your sample, choose and click on any of the 3 filter sets (DAPI, FITC, TRITC buttons) to focus on your sample using the oculars. (You should now see the light coming out of the objective) 5.6) While looking into the oculars, rotate the focus knob to bring the objective up until you see your sample in focus. Be careful not to crash the objective into your sample! Click on the OFF button to turn the light off, then Close the Microscope control window. Important: If the microscope makes a beeping sound when you try to focus. Keep pressing on the button on the right side of the microscope while moving the focus knob to override the focus lock. 5.7) Click on the LSM icon to switch to the confocal imaging mode. 8
6. Loading imaging settings 6.1) Click on the Config button to open the Configuration Control window. 9 6.2) Switch to the Multi Track mode and then click on the Config diskette button to select and then Apply an imaging configuration. (If required lasers are not on, a warning will be shown, see 3.2) Tip: Choose a protocol based on fluorophores present in your sample. For 4 dyes : 405_488_543_633_Fast_Line_Mode protocol is faster than 405_488_543_633_NoBleed_Slow_Frame_Mode, but might have significant cross-talk between the channels! (test by blanking laser lines)
7. Adjusting channels and image parameters 7.1) Click on the Scan button to open the Scan Control window alongside the Configuration Control window 10
7. Adjusting channels and image parameters (cont.) 7.2) Adjusting scan area, image size, pixel depth and scan speed in the Scan Control window. Make sure the Mode button is on Scanning images/2d arrays of pixels - Frame button on. Shows and selects an objective (if new selected refocusing will have to be redone). Mind Immersion! Chose pixel dimensions of your final images by pressing on the preset buttons (1:1 aspect ratio) or by typing in. Start with 512 by 512 or 1024 by 1024. Pixel scan speed, slower the better the signal to noise. Set to 7-9. Pixel depth (# of shades of gray in your image). Choose: 8bit Image averaging for a better signal to noise ratio. The more the longer the scan. Choose: Line Mean 2 or 4 As required: Zoom into the area & rotate the scan area As required: Move the scan area (to center the specimen) 11
7. Adjusting channels and image parameters (cont.) 7.3) Adjusting individual channels for optimal resolution and to minimize under- and overexposed pixels using the Scan Control window. In the Configuration Control window, uncheck all but one track. In the Scan Control window, click on the Channels button Click on the Fast XY button to start fast continuous image scan. An image window will automatically open. Drag to minimize the image window if it overflows the screen. From the menu strip on the right click on the Palette button. In the Color Palette window select Range Indicator and then Close the window. Overexposed pixels will be shown in the image in red, and underexposed in blue. 12
7. Adjusting channels and image parameters (cont.) 7.3) Continued in the Scan Control window Set Pinhole to 1 airy unit by clicking on the 1 button. Estimated Z axis image thickness (Optical slice) will be displayed below. At this point adjust the focus (fine focus knob) to find the brightest focal plane. If necessary, move the stage to find another area. If there are many blue pixels, increase the Offset. If there are no blue pixels decrease the Offset until you see very few blue pixels. If the image is dim (dark gray/black) increase Detector Gain and/or Laser Power (next to the checked box) until you see few red pixels. Sometimes, if staining is too weak, image brightness can be increased by slightly opening the pinhole. If the image has red pixels reduce Laser Power (next to the checked box) and/or Detector Gain. Important: the Laser Power slider for the 405 diode laser is not operational, adjust Detector Gain only! SEE LAST PAGE FOR MORE Click on the Stop button to stop the scan. Too many blue pixels & low signal. Solution: 1. Focus better 2. Increase amplifier offset to eliminate blue pixels 3. Increase laser power or detector gain (to brighten pixels up). Too many blue pixels & high signal. Solution: 1. Focus better 2. Increase offset to eliminate blue pixels 3. Decrease laser power or detector gain (to desaturate pixels up). Well-adjusted channel: No blue, no red pixels. Pixels are well stretched in the histogram. 13
8. Taking a snap 8.1) Go back to the beginning of 7.3 step to adjust the remaining tracks one by one. 14 In the Configuration Control window, uncheck the adjusted track and check the next on to be adjusted. 8.2) After all of the tracks are adjusted. Check all of the tracks. In the Configuration Control window check all of the tracks. 8.3) To take a snap of your sample with all channel. Click on the Single button in the Scan Control window. Save the image in your folder. (tip: if images are grey in color change the palette to no palette, see p.12) 8.4) In the image window: To show a merge of all channels click on the xy button, to show all channels individually click on Split xy button. To draw a scale bar, click on the Overlay button, select the scale bar and draw it on the image. To adjust image contrast and brightness click on the Contr button. You can reuse all of your settings next time you come to use the microscope by opening a past image and clicking on the reuse button!
9. Taking a Z-stack Source for the slide: Zeiss 510 Meta Tour Presentation 15
10. Shutting the microscope down 1. Save and close all of your images 2. Click on the laser button and turn all of the lasers OFF 3. Close the AIM program 4. Copy your data to your USB storage device 5. Turn off the computer 6. Remove your sample from the microscome stage and wipe the objective clean with LENS PAPER 7. Cover the microscope with the dust cover 8. If 5 minutes have not yet passed since turning off the lasers, wait, then turn the remote control OFF (switch #3) 9. Turn the mercury lamp OFF (switch #1) 16
Only If needed: the power of the 405 diode laser can be changed by the adjustment knob on the laser driver box as shown below: 17