SHORT GUIDE TO LASER MICRODISSECTION USING THE PALM COMBI SYSTEM

Transcription

1 SHORT GUIDE TO LASER MICRODISSECTION USING THE PALM COMBI SYSTEM Turning ON the PALM DuoFlex Combi system 1. Turn on the three power point switches on the wall. From right to left: mercury lamp, microscope and PALM DuoFlex Combi system including heater. If need to use heater for effective temperature control, make sure heating unit and TempModule are both on (see right). Check switches at back of both of these units. 2. If you have fluorescent samples and need to use fluorescence imaging to identify and capture samples then (and only then!) turn on the X- Cite halide lamp. Lasts 2000 hours. See right. 3. Turn on the AxioObserver microscope at the LHS of the microscope. TFT Display (Touch screen) will come on. 1

2 4. Switch the key clockwise to turn on the PALM control box. The system should show many green lights that means the system is working well. 5. Turn on the computer. Log in using Zeiss. There is no password 6. Enter the PALM Robo software V4.5. The program layout will be revealed. 2

3 7. To deselect optical tweezer laser markers (green and red calipers), select View, then Laser Marker. Select appropriate laser marker in pull down menu and deselect Show Laser Marker. Then click OK. Operation of PALM Robo software for LMPC of samples on slides only 1. To load slides: Select load position icon on the menu toolbar. The stage will physically slide away from the objective lens to allow loading of the slide into the slide holder. The Slide holder can hold up to 3 slides at one time. Place membrane and/or sample facing upwards. 3

4 NOTE: The slide holder only goes in one way. Note the upper right hand corner of the slide holder has a right- angled edge. When slide holder in position select Return to working area in software 2. Click on the navigator icon on the top menu. This opens the PALM Navigator Window. Using the PALM Navigator you can scan your slide or certain parts of it and easily move the stage to points defined by a mouse click. 3. Choose the correct objective lens in the Microscope window and set up for transmitted light or reflected light (fluorescence imaging). Always start with a low objective and work upwards. 4. Choose the correct camera: ICc for laser microdissection (LHS) and and AxioCam MRm Rev3.SN: 3430 (LHS) for fluorescence imaging. NB. The fluorescent signal may be lost rapidly when imaging with the ICc camera and it is best not to cut using the HRm camera. 4

5 5. To acquire a live image: Select Auto- live to acquire exposure time automatically or deselect Auto- live and select Measure to increase or decrease the exposure time manually. Adjust the Gain if you need to (increase the gain and reduce exposure time in order to reduce bleaching). 6. Calibrate the position of the laser every time you use the system and for each objective: Using a test slide (eg. membrane- coated slide) or area of tissue not needed. Select Calibration, then Cut Laser Adjustment Wizard. The wizard will instruct you how to proceed: 5

6 Rough adjustment of energy: Select best cutting performance eg. #7 6

7 Rough adjustment of focus: Select best cutting performance eg. #15 If needed (recommended), do a fine adjustment of energy and focus also. Finally set position of cut laser marker: Put target over where cut laser is (shown by dot) 7

8 Calibration is complete. Some energy adjustment may need to be made with your sample 7. Check that both the cutting and LPC settings are working well on an area of tissue AWAY from the area to dissect and LPC. 8

9 You may need to play a little more with both the cutting and LPC settings to get this right for your sample at the objective you are using. NB. The laser cuts at the sample plane so make sure the laser is in the correct focus position. One can also adjust the distance of auto LPC shot dots (eg. 5) and distance of auto LPC shot dots from line (eg. 3) using the Robo software. Save your settings so that you can use these for similar samples next time. 8. When you are ready to collect your LPC samples. Click on the Capture Device icon to change collector. Select the most appropriate collector. The SingleCap/Single Tube collectors are the most commonly used. One has a diffusor (see arrow) for improved imaging. NB. If not using sticky caps, add 40-50µl collection medium only, eg. DEPC- treated water to the cap before loading the cap sideways on the collector plate (as shown). The tube lies horizontally so that no sample can be inadvertently catapulted into the tube. 9. Load collector carefully onto the Robomover platform (see right). Do not put weight on holder. Select Scan new collector type in software. 9

10 The Robomover will move the collector and read off its barcode so that it can reveal a picture of the collector in the software. You can click on the diffusor or cap to move these areas over the objective. 10. When all the settings are optimized and ready for catapulting, select the cap in Robo mover to load the cap into position. The cap is now positioned over the sample that is ready to LPC. If the cap is touching the sample, you can move this up in the software. If the cap is too far away from the sample, then you can move the cap down. 11. To observe fluorescent staining, set up reflected light and choose the appropriate filter (DsRed, FITC or DAPI). Select the ICC camera for LPC. (NB. do not change cameras during this process). Find the cells of interest. You can freeze the image, so that you can select your elements to draw without bleaching your sample further. You will need to unfreeze the image to play with your laser and/or TL settings. You are ready now to cut and LPC your samples. 12. To check that you have collected LPC sample in the cap: Click to observe the cap under microscope. You will most likely need to change to a low objective to locate the cap and sample first. You can then move to a higher objective. Select to go back to point of origin. 10

11 13. Save images as you go or automatically before and after LPC using the software. 14. Save your elements list if you need to keep track of these. Turning off the PALM system 1. Remove the cap collector. Remove your tubes and store them appropriately. Put sample collector back in the appropriate blue box. 2. Remove the slide holder. Remove slides and place slide holder back on stage in place. 3. Change back to the lowest objective lens in the software. 4. Save all images. 5. Exit the PALM Robo software. 6. Shut down the computer. Wait for this to shut down completely. 7. Turn off Zeiss system by switching the key anti- clockwise. 8. Turn off the microscope. 9. Turn off the mercury lamp. 10. Turn off three switches on the wall from left to right. 11. Place the cover on the microscope. 12. Log your usage in the log book. 13. Leave the room clean and tidy. 14. Switch off the A/C and lights. 11