Zeiss Axio-Observer spinning-disk microscope

J-A Bombardier Building, Room 3223-011 

Advanced Microscope Tier 2 usage price

Instrument awarded to Dr. Daniel Zenklusen by the Canadian Foundation for Innovation (CFI)

• Light sources

  • 12V 100W halogen lamp LED for transmitted light
  • Lumencor SOLA for visible fluorescence
    

    Développer
    title Lumencor SOLA complete specifications:
    Emission peak (nm) Power (mW) 334 5 365 20 405 19 436 22 546 21 579 18

  •  4 4x lasers: 405, 488 561 638
    

    Développer
    title Laser complete specifications:
    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

  1. 100x/1.46 Oil WD 0.11
  2. TIRF Divergence adjusting aid
  3. TIRF Angle adjusting aid 
  4. 63x/1.46 Oil WD 0.1
  5. 10x/0.3 Air WD 5.2
  6. Empty

BF: Bright-field
DIC: Interference contrast

1. Turn on the computer (#1)
2. If required, turn on the incubation power bar

3. Turn on the camera and laser power bar on the left of the microscope (#2)

4. Turn on the microscope power bar on the right of the microscope (#3)

5. Use your UdeM credentials to log in to Windows
   

   Remarque
   When using for the first time, it is necessary to import the microscope-specific parameters BEFORE starting the software. See the First Use section below.

6. Start the Zen Blue software

When using for the first time, it is necessary to import the microscope-specific parameters into the software. This procedure is usually carried out during the training session.However, it is also possible to use it to reset the software if it is not displayed correctly, for example.

1. If open, close the Zen Blue software and wait for it to close completely (up to 30 seconds)
2. On the Desktop open the Documentation folder
3. Double-click Settings for Axio-Observer Z1
4. A script will run and a black window will appear briefly
5. You can then reopen the Zen Blue software

This procedure puts the microscope in a safe configuration and performs a focus calibration. At the end of this procedure the microscope will be ready for acquisition.

• Files can be saved temporarily (during acquisition) 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).

1. Save your data
2. Close the software Zen Blue
3. Transfer your data to the D: drive (Data Storage) or to your external drive and delete it from the local C: drive
4. If used, turn of the incubation power bar
5. If used, clean oil lenses with lens cleaner and paper

6. Turn off the microscope power bar on the right of the microscope (#3)

7. Turn off the camera and laser power bar on the left of the microscope (#2)
8. Turn off the computer

• 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).

• 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 
• NOTE: AOTF2 for FRAP

• 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)

• 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

The A spinning disk is a multipoint confocal.Outside is a multipoint confocal: due to the confocality, signal is restricted to the focal plane. Outside of the focal plane, the sample disappears completelyis not visible. It is therefore thus easier to find your sample through the eyepieces.Follow the tuning procedure to achieve thisthe proper focal plane using the oculars (wide-field signal). Please refer to first focus" procedure on the "loading samples" tab.

The best way to solve a problem in Microscopy is to follow the light path. You will find in the Light path tab of this page, the diagrams which will allow you to follow the light all along its path through the microscope.

1. Open the light path file
2. Starting from the light source and moving towards the detector, verify that there is indeed light after each component of the microscope

Yes. It is an inverted microscope designed for the observation of living specimens. The spinning disk is particularly appreciated for its limited phototoxicity and low out-of-focus background. This is an inverted microscope designed to look at specimen in a dish or a multi-well plate. The objectives are optimized to image through thin glass bottom multi-well plates. You may also image specimen mounted between a slide and a 0.17mm thick coverslip.