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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)

Main 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

    • 4x 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

    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

Position

Name

Brand

Complete Name

Identifier

Magnification

Numerical Aperture

Immersion

Type

Working distance (mm)

Transmittance
(% [nm])

Techniques

Coverslip thickness (mm)

1

100x/1.46 Oil

Zeiss

100x/1.46 DIC I
Alpha Plan-ApoChromat
M27

420792-9800

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
Alpha Plan-Apochromat Korr TIRF
M27

420780-9970-000

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

420340-9901

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:

    1. Empty
    2. BS_455
    3. LSM TFT80/20 1447-381
    4. BF RL TIRF Calibration 424928
    5. Set 76 C/G/Dr
    6. Set 77 G/R/A6
PositionNomMarqueIdentifiantFiltre d'excitation Miroir dichroïqueFiltre d'émissionCommentaires
1DAPI
Filter Set 49		Zeiss

488049-9901

365/50
[325-375]		395LP445/50
[420-470]
  • 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
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.
  • Start the Zen Blue software
  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 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:

  1. Press Home>Load Position to lower the stage to its lowest position
  2. Press Set Work Position to store this position
  3. If necessary, move the focus slightly up to remove the "Lower Z limit reached" message displayed on the touchscreen
  4. Press Home>Microscope>Turret>Objectives>10x to select the 10x objective
  5. If asked, tap Done to remove the oil lens cleaning warning
  6. Press Home>Microscope>XYZ>Position>Z-Position>Set zero>Auto to perform focus calibration
  7. Press OK to start the focus calibration procedure
  8. 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

Make sure to calibrate the focus before performing the first focus.

On the microscope touch screen:

  1. Press Home>Microscope>Turret>Objectives
  2. Press 10x to select the 10x lens

why 10x ?

The 10x objective is the safest because it has the longest working distance (5.3 mm). The sample will appear perfectly sharp long before the lens approaches it. It is recommended to always first focus with the safest lens. The objectives are para-focal: focusing with the safest objective will then allow you to easily find your sample with another objective.
  1. Press Home>Load Position to lower the stage to its lowest position
  2. Press Set Work Position to store this position
  3. If necessary, move the focus slightly up to remove the "Lower Z limit reached" message displayed on the touchscreen
  4. Place the test slide on the microscope stage with the coverslip toward the objective
Always use the test slide to perform the first focus.
  1. If necessary, move the stage so that the sample is centered on the objective

On the computer:

  1. Open the Zen Blue software
  2. In the Locate tab, select BF or the desired fluorescence ((CFP/GFP/DsRed ou GFP/RFP/Cy5, etc…) to activate the configuration
  3. 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

Important: First focus with the safest lens (10x) before selecting another lens and continuing with secondary focus (see section "Primary Focus" above).

no Air Objectives on this instrument

This microscope only has a 10x air objective (see section "Primary Focus" above). However, if there were other, higher magnification objectives requiring air as a media, the procedure would be as follows:

After performing the first focus, on the microscope touch screen:

  1. Press Home>Microscope>Turret>Objectives
  2. Press on the desired lens

In Zen Blue software:

  1. In the Locate tab, select BF or the desired fluorescence (CFP/GFP/DsRed ou GFP/RFP/Cy5, etc…) to activate the configuration
  2. Adjust the focus with the precision dial while looking through the eyepieces until the image is perfectly sharp
  3. In the Locate tab, select Off to turn the illumination off
  4. Your sample is ready for acquisition!

After performing the first focus, on the microscope touch screen:

  1. 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.

The 63x objective is a very good objective because it has the a good transmittance and a larger field of view than the 100x. Given the pixel size of the camera, Nyquist sampling can reach the resolution limit when using the 1.6x tubelens.
  1. Remove your sample from the microscope
  2. Place a drop of oil on the objective
  3. Replace your sample from the microscope
  4. Press Done. The microscope will automatically return the sample to its original position

In Zen Blue software:

  1. In the Locate tab, select BF or the desired fluorescence (CFP/GFP/DsRed ou GFP/RFP/Cy5, etc…) to activate the configuration
  2. Adjust the focus with the precision dial while looking through the eyepieces until the image is perfectly sharp
  3. In the Locate tab, select Off to turn the illumination off
  4. 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.

  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


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).
  • 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 
  • 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


Troubleshooting

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

FAQ

Yes. This is an inverted microscope, designed to observe living samples. The spinning disk microscope is notably relied upon thanks to its lower phototoxicity and low out-of-focus background.

Plus de dépannage et de FAQs sont disponibles dans la version anglaise.


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