Olympus FV3000 Manuel utilisateur

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Olympus FV 3000 operation manual

1. Switch on PC (password olympus)

2. Switch on the main power switch on the cord extender in the Rack. Stage control

and microscope controller will switch on automatically.

3. Switch on laser lines which you plan to use. System has 405nm, 445nm, 488nm,

514nm, 561nm, 594 nm and 640 nm cw diode lasers.

4. Turn on the key to activate the lasers. The lasers will blink with green. Then they

show white lamp on, they are ready to use.

5. You can add a laser line later when software is running too.

6. Turn on touch PC (the switch is on the back) and wait until it is initialized. Blue and

green light should light up on the controller.

7. If you need heating and CO2, switch on the cord extender for the Cell Vivo (heating

and CO2 control unit) and start Cell Vivo software. And start heating 1hour in

advance minimum

8. Start the FV31S-SW software

9. Now you can switch on FLIM system (use a FLIM guideline)

10. Keep resonant scanner unchecked if you do not need it. If you need it, for stability

start it 30 minutes before your scans.

11. Keep other settings in the start dialog unchanged (enable XY on)

12. Choose NO for stage cleaning

13. Choose objective either in software or on touch PC. The microscope has 1.25x NA

0.04, 4x NA 0.16, 20x NA 0.8, 40x NA 1.4 oil, 30x NA 1.05 Silicon oil and 60x NA 1.3

Silicon oil. If using oil objective, CHECK THAT YOU USE RIGHT TYPE OF OIL

14. Check the collar correction on the objective. It should be set to 0.17mm if you use

1.5# cover slips. Note that at 23C and at 37C the correction is different.

15. Use the Camera/Eye button on the touch panel controller to switch between Laser

Scanning Mode (LSM, camera symbol) to Ocular view (eye symbol). You will probably

need to adjust light intensity.

16. Find your sample using epifluorescence (EPI) or transmission light (DIA). For EPI

select Quad filter, on position. 2.

17. For EPI choose 1V for violet excitation (DAPI), 2B for blue excitation (GFP) and 3GR

for red excitation (RFP) for LEDs.

18. Open and close shutter when needed

19. Check that COOL LED is on.

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20. In software choose in Ocular pannel, and LSM

21. In the “LSM imaging “panel adjust adjust Scan

Setting: Scanner Type: Galvo, Oneway mode. Scan

speed: 2µs/pix.

22. In the “PMT” panel adjust Scan Setting: use VBF.

23. In the “PMT” panel set channels for your dyes, by pressing the “Dye & Detector

select” button. Adjust emission bandwidth if needed.

24. In the “PMT” panel check the confocal aperture. Use AUTO to have it averaged for all

channels.

25. Start with low laser power 0.5%, HV 500, offset 0% (max 3%), do not change gain.

You can set up two 4 channels in one group plus transmission light channel.

Detection regions should not overlap with laser lines.

26. In the “Area Settings” panel select the Scan Size and use the Optimize button to

adjust the Zoom for Nyquist-sampling

27. Check for over and underexposure (Hi-Lo).

28. Check bleadthrough, i.e if you see signal from one laser in another channel. If

bleadthrough, go to “PMT” panel, select “sequential sc” and the mode “Line” or

“Frame” (Frame is faster).

29. Be careful and do not overexpose PMTs. (indicated by red colour in Hi-Lo mode)

30. Set a folder where your images will be saved. Use the D-drive for it and find your AG-

folder. The image format will be *.oir

31. Set averaging/accumulation if you need it.

32. Check if Z series and t-series are off

33. Do your imaging: press LSM-START

Finish your work

1. When finished: close the software.

2. if used heating and CO2 then stop heating and CO2 in the Cell Vivo software and

close it. Switch off Cell Vivo switch on the power cord. Close CO2 valves.

3. Copy your data to the data transfer server.

4. Switch off FLIM system if it was on

5. On the touch PC screen press OFF, when OLYMPUS is displayed, switch off the touch

PC using the switch on the back.

6. Turn the key to deactivate lasers.

7. Switch off all lasers which are on.

8. Switch off the main switch on the power cord in the rack.

9. Remove your sample

10. Clean oil objective with isopropanol and lens paper.

11. Write you name, date and comments in the electronic log book.

12. Switch off the PC.

For Z-stack:

1. In acquisition choose Z-series on (Z Section panel, bottom right)

2. Find bottom position, register it, then find a top position and register it.

3. Go from the lower position to higher position.

4. Optimize step size accordingly to your needs (Or press the “Optimize” button).

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ZDC: drift compensation (does not work with 1.25x and 4x)

1. Check the current position and check NEAR-limit settings: approx. numbers 1800 for

cover slips, petri dish 2300, well plate about 3500. (Please verify the values for petri

dish and well plate with your vessels again)

2. Press “Apply entered parameters to near limit”.

3. Place ZDC dichroic mirror in: ZDC DM IN

4. On Touch PC press START for focus search in EPI mode. If it is successful, you hear

one beep.

5. Use Single shot mode for multipoint imaging experiments (Z stacks etc) and

Continuous mode for long-term time-lapse experiments.

6. At ZDC you cannot change objectives.

7. ZDC does not work well with oil objective, but good with Silicon oil objective.

Objectives:

30x Silicon and 60x Silicon are good for live cell imaging: SILICON OIL

40x oil is good for fixed samples: NORMAL OIL

Shortcuts:

1. F11 start/stop live

2. F12 start image scan

3. Ctrl and mouse scroll: focusing

4. Double click on the image while life moves the stage

Provisionary

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OIL:

To use Cell Vivo heating and CO2 controller:

1. If you did not start it before press the button on the power extender under the Cell

Vivo controller.

2. Start Cell Vivo software.

3. To heat the incubator set the temperature H unit 2000 to T you need and press ON.

4. You can log Temperature data.

5. To have stable temperature 1-2 hours heat up time is required.

6. When finished, stop heating

7. Close the software

8. Switch off the controller using the button on the power extender.

Stage control

40x oil

30x and 60x

Silicon oil

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