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Zeiss Axio-Observer spinning-disk microscope
J-A Bombardier Building, Room 3223-1
Advanced Microscope Tier 2 usage price
Instrument awarded to Dr. Daniel Zenklusen by the Canadian Foundation for Innovation (CFI)
- Applications
- Transmitted light
- Fluorescence
- Live-cell imaging
- Incubator (Temperature & CO2)
- Light sources
- LED for transmitted light
- Lumencor SOLA for visible fluorescence
Emission peak (nm)
Power (mW)
334
5
365
20
405
19
436
22
546
21
579
18
- 4 lasers 405, 488 561 638
Laser line (nm)
Nominal Power (mW)
Power at sample plane in mW
(Obj. 10x; 2024/07/02)
405
50
0.7
488
100
3.3
561
50
2.3
639
50
1.1
- Objectives
- 100x/1.46 Oil WD 0.11
- TIRF Divergence adjusting aid
- TIRF Angle adjusting aid
- 63x/1.46 Oil WD 0.1
- 10x/0.3 Air WD 5.2
- Empty
Position | Name | Brand | Complete Name | Identifier | Magnification | Numerical Aperture | Immersion | Type | Working distance (mm) | Transmittance | Techniques | Coverslip thickness (mm) |
1 | 100x/1.46 Oil | Zeiss | 100x/1.46 DIC I | 100x | 1.46 | Oil | Plan Apochromat | 0.11 | >70% [410-800] | BF, DIC, Fluo | 0.17 | |
2 | TIRF Divergence adjusting aid | Zeiss | 423682-8801-000 | |||||||||
3 | TIRF Angle adjusting aid | Zeiss | 423682-8802-000 | |||||||||
4 | 63x/1.46 Oil | Zeiss | 63x/1.46 DIC III | 63x | 1.46 | Huile | Plan Apochromat | 0.10 | >80% [500-800] | BF, DIC, Fluo | 0.15-0.19 | |
5 | 10x/0.3 Air | Zeiss | 10x/0.3 EC Plan-NeoFluar | 10x | 0.3 | Air | Plan-NeoFluar | 5.2 | >90% [450-750] | BF, DIC, Fluo | 0.17 | |
6 | Empty |
BF: Bright-field
DIC: Interference contrast
- Filter cubes:
- Empty
- BS_455
- LSM TFT80/20 1447-381
- BF RL TIRF Calibration 424928
- Set 76 C/G/Dr
- Set 77 G/R/A6
Position | Nom | Marque | Identifiant | Filtre d'excitation | Miroir dichroïque | Filtre d'émission | Commentaires |
---|---|---|---|---|---|---|---|
1 | DAPI Filter Set 49 | Zeiss | 365/50 [325-375] | 395LP | 445/50 [420-470] |
- Detector
- 2 EMCCD camera Photometrics Evolve 512 x 512 pixels, 16-bit, 33 f/s at full resolution, sensor size 8.192 mm x 8.192mm, pixel size 16um x 16um Evolve512-Datasheet.pdf Evolve512_Manual.pdf
- Turn on the computer (#1)
- If required, turn on the incubation power bar
- Turn on the camera and laser power bar on the left of the microscope (#2)
- Turn on the microscope power bar on the right of the microscope (#3)
- Use your UdeM credentials to log in to Windows
- Start the Zen Blue software
- If open, close the Zen Blue software and wait for it to close completely (up to 30 seconds)
- On the Desktop open the Documentation folder
- Double-click Settings for Axio-Observer Z1
- A script will run and a black window will appear briefly
- You can then reopen the Zen Blue software
This 2-minutes procedure puts the microscope in a safe configuration to avoid mishaps with the objectives or samples.
Thank you in advance for following this guideline every time!
On the microscope touch screen:
- Press Home>Load Position to lower the stage to its lowest position
- Press Set Work Position to store this position
- If necessary, move the focus slightly up to remove the "Lower Z limit reached" message displayed on the touchscreen
- Press Home>Microscope>Turret>Objectives>10x to select the 10x objective
- If asked, tap Done to remove the oil lens cleaning warning
- Press Home>Microscope>XYZ>Position>Z-Position>Set zero>Auto to perform focus calibration
- Press OK to start the focus calibration procedure
- Wait a few seconds for the calibration to be completed
Once calibrated, the focus can be found at Z = 5 mm). The Z value can be found on the microscope touch screen Home>Z-Position
On the microscope touch screen:
- Press Home>Microscope>Turret>Objectives
- Press 10x to select the 10x lens
why 10x ?
- Press Home>Load Position to lower the stage to its lowest position
- Press Set Work Position to store this position
- If necessary, move the focus slightly up to remove the "Lower Z limit reached" message displayed on the touchscreen
- Place the test slide on the microscope stage with the coverslip toward the objective
- If necessary, move the stage so that the sample is centered on the objective
On the computer:
- Open the Zen Blue software
- In the Locate tab, select BF or the desired fluorescence ((CFP/GFP/DsRed ou GFP/RFP/Cy5, etc…) to activate the configuration
- Adjust the focus with the main dial while looking through the eyepieces until the image is perfectly sharp
Once calibrated, the focus can be found at Z = 5 mm). The Z value can be found on the microscope touch screen Home>Z-Position
In the Locate tab, select Off to turn off the illumination
no Air Objectives on this instrument
After performing the first focus, on the microscope touch screen:
- Press Home>Microscope>Turret>Objectives
- Press on the desired lens
In Zen Blue software:
- In the Locate tab, select BF or the desired fluorescence (CFP/GFP/DsRed ou GFP/RFP/Cy5, etc…) to activate the configuration
- Adjust the focus with the precision dial while looking through the eyepieces until the image is perfectly sharp
- In the Locate tab, select Off to turn the illumination off
- Your sample is ready for acquisition!
After performing the first focus, on the microscope touch screen:
- Press Home>Microscope>Turret>Objectives
2. Press 63x Oil (1.4) or 100x Oil (1.4) to select the desired lens. The microscope will automatically lower the stage so that the sample is accessible.
- Remove your sample from the microscope
- Place a drop of oil on the objective
- Replace your sample from the microscope
- Press Done. The microscope will automatically return the sample to its original position
In Zen Blue software:
- In the Locate tab, select BF or the desired fluorescence (CFP/GFP/DsRed ou GFP/RFP/Cy5, etc…) to activate the configuration
- Adjust the focus with the precision dial while looking through the eyepieces until the image is perfectly sharp
- In the Locate tab, select Off to turn the illumination off
- Your sample is ready for acquisition!
Storage management
- During an acquisition session, files can be saved temporarily on the local C: drive (desktop)
- At the end of each session, copy your data to your external drive and delete it from the local C: drive
- You can store your files on the D: drive (Data Storage). If you do, please create a folder per laboratory using the principal investigator last name. Within, create one folder per user (Firstname_Lastname).
In any case, your files should be removed from the C: drive at the end of your session.
- Save your data
- Close the software Zen Blue
- Transfer your data to the D: drive (Data Storage) or to your external drive and delete it from the local C: drive
- If used, turn of the incubation power bar
- If used, clean oil lenses with lens cleaner and paper
- Turn off the microscope power bar on the right of the microscope (#3)
- Turn off the camera and laser power bar on the left of the microscope (#2)
- Turn off the computer
Reminder
- Take back your samples including those inside the microscope chamber
- Leave the microscope and the working area clean
- 488nm laser power loss: alignement
(FIXED) Problem with the FRAP module:
At first could not modulate the laser intensity (yet could not find the spot on the field), rebooted and then the FRAP fiber does not carry any light (even 488)
==> Laser manipulation module was too far in x and y (noticeable as a tilt and a gap between both parts of the module).
Corrected Trigger Module configuration for the BackCam
- reassigned each camera with its own trigger module in MTB config
- DualCam calibration parameters set, using beads
- Realigned Lasers:
- Laser output measured in spinning disk mode at the sample with 10x objective, 1x Extended tubelens, 100% laser power and ALL lasers ON:
- 405nm: 0.70 mW before => 0.66 mW after
- with laser 488 OFF: 0.81 mW before => 0.83 mW after
- 488nm: 3.75 mW before => 4.17 mW after
- with laser 405 OFF: 3.95 mW before => 4.33 mW after
- 561nm: 2.1 mW before => 2.37 mW after
- 639nm: 0.9 mW before => 1.1 mW after
- 405nm: 0.70 mW before => 0.66 mW after
- Laser output measured in spinning disk mode at the sample with 10x objective, 1x Extended tubelens, 100% laser power and ALL lasers ON:
- NOTE: AOTF2 for FRAP
AOTF2 calibration
For FRAP, the illumination is strongest with the AOTF2 at 60% and not 100%
· Error message when switching between FRAP & SD illumination
AOTF2 Error message
FRAP/SD illumination switching triggers an error message " 'MTBAOTF2LaserLine6' is not supported by the current hardware"
- Zen 2.6 updated hotfix 12
- Microsoft Windows update
- Laser output measured in spinning disk mode at the sample with 10x objective, 1x Extended tubelens, 100% laser power and ALL lasers ON:
- 405nm: 1.08 mW StdDev 0.01mW
o 488nm: 3.3 mW StdDev <0.01mW (3.7 mW if laser 405 is OFF)
405nm and 488nm Interactions
When both 405nm and 488nm laser are ON simultaneously, the output power is decreased by 11%.
- 561nm: 2.6 mW StdDev <0.01mW
- 639nm: 1.37 mW StDev 0.02 mW
· Control for vibrations during construction work on the 4th floor
- Error when the definite focus is used (unexpected error the definite focus did not respond)
- To solve it:
- Turn off the Zen Software
- Turn off the microscope power bar (#3)
- Turn off the definite focus by pressing and holding the definite focus button
- Turn on the definite focus by pressing once the definite focus button
- Wait until the definite focus display shows "Detecting stand"
- Turn on the microscope power bar (#3)
- Confirm that the definite focus is showing in the microscope tactile display
- Open Zen software and run an experiment using the definite focus to confirm it is fully functional
Added to wiki
- AOTF1 changed
- Aucune étiquette