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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
The following diagrams allow you to follow the light path in transmitted light (bright field, DIC) and in reflected light (fluorescence).
To do
Check Loss of 488nm laser power
2024-04-26
(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).
2024-03-04
Corrected Trigger Module configuration for the BackCam
- reassigned each camera with its own trigger module in MTB config
2024-02-29
- 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"
2021-11-20
- 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
2021-11-03
- 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
2021-09-27
- Added to wiki
2020-07-07
- AOTF1 changed
- Aucune étiquette