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Zeiss Axio-Observer Z1 inverted microscope

Roger Gaudry Building, Room R-421
Simple Microscope usage price

Instrument awarded to Dr. Audrey Claing and Jean-Philippe Gratton by the Canadian Foundation for Innovation (CFI)

  • Applications
    • Bright-field
    • Phase contrast
    • Fluorescence

  • Light sources

    • LED lamp for transmitted light

    • Colibri 7 (385/469/555/631) for fluorescence

      Emission peak (nm)

      		Power (mW)
      385/30150
      469/38110
      555/3040
      631/3350

      Information Zeiss Colibri 7


  • Objectives

    1. 2.5x/0.085 Air WD 8.8

    2. Empty

    3. 10x/0.25 Air Ph1 WD 6.5

    4. Empty
    5. 20x/0.5 Air Ph2 WD 2.0
    6. 40x/0.95 Air WD 0.25
      PositionNameBrandFull nameIDMagnificationNumerical ApertureImmersionTypeWorking distance (mm)Transmittance
      (% [nm])      		TechniqueCover glass thickness (mm)
      120x/0.5 AirZeiss20x/0.50 Ph2
      EC Plan-Neofluar
      M27      		420351-991020x2.0AirPlan Neofluar2.0Not AvailableBF, PhC, Fluo0.17
      2

      Empty












      3

      40x/0.95

      		Zeiss40x/0.95
      Plan-Apochromat Corr
      M27      		420660-9970

      40x

      		0.95

      Air

      		Plan ApoChromat0.25>80% [400-840]BF, Fluo

      0.13 - 0.21
      Correction ring

      4Empty










      52.5x/0.085 AirZeiss2.5x/0.085
      EC Plan-Neofluar
      M27       		420320-99022.5x0.085AirPlan Neofluar8.8>95% [400-750]BF, Fluo0.17
      610x/0.25 AirZeiss10x/0.25 Ph1
      N-Achroplan
      M27       		420941-991110x0.25AirAchroPlan

      6.5

      		Not availableBF, PhC, Fluo0.17

  • Filter cubes
    1. DAPI
    2. GFP
    3. Rhodamine
    4. DHE (dihydroethidium)
    5. Cy5
    6. Quadruple DAPI/GFP/Cy3/Cy5
      PositionNameBrandIDExcitation filterDichroic mirrorEmission filterComments
      1DAPI
      Filter Set 49      		Zeiss

      488049-9901

      		365/50
      [325-375]      		395LP445/50
      [420-470]
      2GFP
      Filter Set 13      		Zeiss488013-0000

      470/20
      [460-480]

      495LP

      		517/25
      [505-530]
      3Rhodamine
      Filter Set 43      		Zeiss

      000000-1114-101

      		545/25
      [533-567]      		570LP605/70
      [570-640]
      4DHECustom

      Custom

      500/50
      [475-525]

      		540LP580/20
      [570-590]      		Undefined specifications
      Best guess values
      5Cy5
      Filter Set 50      		Zeiss488050-9901640/30
      [625-655]      		660LP

      690/50
      [665-715]


      6Quadruple
      DAPI/GFP/Cy3/Cy5
      FS90 HE LED
      		489090-9110-000


      		QBS 405 + 493 + 575 + 653

      QBP 425/30+514/30+592/25+709/100

      		Excitation filters included in the light source FS90 HE LED
  • Detector
    • Zeiss AxioCam MR R3 CCD Camera 1388 x 1040 pixels, 12-bit, 13 images/s at full resolution, detector size 8.9 mm x 6.7 mm
Startup

  1. Remove the dust cover from the microscope

  2. Turn on the computer (#1)

  3. If necessary, turn on the power bar for the incubation module (#2A) and open the CO2 cylinder (#2B)

  4. Turn on the power bar at the left of the computer monitor (#3)

  5. Press the microscope start button located on the rear left of the microscope (#4)

  6. Log in Windows using your UdM credentials

    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.

  7. Start the Zen software
First Use

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.

Please note, this procedure will delete all your experiment protocols and restore the software to its original settings.

  1. If open, close the Zen 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-Osberver Z1
  4. A script will run and a black window will appear briefly
  5. You can then reopen the Zen software
Loading samples

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.

Focus Calibration Z

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 = 1.5 mm). The Z value can be found on the microscope touch screen Home>Z-Position

First focus

Important

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

    The 10x objective is the safest because it has the longest working distance (6.5 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.

  3. Press Home>Load Position to lower the stage to its lowest position
  4. Press Set Work Position to store this position
  5. If necessary, move the focus slightly up to remove the “Lower Z limit reached” message displayed on the touchscreen
  6. Place the test slide on the microscope stage with the coverslip toward the objective

    Important

    Always use the test slide to perform the first focus.

  7. If necessary, move the stage so that the sample is centered on the objective

On the computer:

  1. Open the Zen software
  2. In the Locate tab, select BF or the desired fluorescence (GFP, DsRed, DAPI, 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 =  1.5mm). The Z value can be found on the microscope touch screen Home>Z-Position


  4. In the Locate tab, select Off to turn off the illumination
Seconday focus

Important

First focus with the safest lens before selecting another lens and continuing with secondary focus.

Focusing with air objectives

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

  1. Press Home>Microscope>Turret>Objectives
  2. Press 20x or 40x to select the desired lens

    The 40x objective is the best Air objective because it has the greatest number of optical corrections (Plan Apochromat) and the largest numerical aperture (0.95). It offers a lateral resolution of 420nm at a wavelength of 550nm.


  3. Adjust the focus with the precision dial while looking through the eyepieces until the image is perfectly sharp
  4. Your sample is ready for acquisition!
Focusing with oil lenses

This microscope does not have oil immersion objectives. However, if there were, the procedure would be as follows:

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

  1. Press Home>Microscope>Turret>Objectives

  2. Press 63x Oil, 100x Oil (1.3) ou 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 the best oil objective because it has the most optical corrections (Plan Apochromat) and the largest numerical aperture (1.4). It offers a lateral resolution of 240nm at a wavelength of 550nm.

  3. Place a drop of oil on your sample
  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 (GFP, DsRed, DAPI, 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
  • 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).

In any case, your files should be removed from the C: drive.

Shutdown
  1. Save your data
  2. Close the Zen software
  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 off the incubation module power strip (#2A) and close the CO2 cylinder (#2B)

  5. Select the 10x objective and press load position to bring the objectives to the bottom position

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

  7. Turn off the computer
  8. Cover the instrument with the protective dust cover

Important Reminders

  • Take back your samples including ones in the microscope
  • Leave the microscope and the working area clean

The following schematics depict the light path for transmitted (bright-field and Phase Contrast) and reflected (fluorescence) lights.

To do
  • Check stage tilt
  • Ask for Colibri Remote
  • Ask for fluorescence backlight from LED
2024-02-22 Microscope Firmware update
  • Microscope Firmware update to add Colibri to the touchscreen
  • Zen 3.5 HotFix 10
2022-05-09
  • Computer replacement
  • Added Colibri
  • Added startup procedure
  • Parafocality and paracentrality
2022-03-17
  • Added complete description
2021-09-27
  • Added to wiki

Stand

  • Zeiss Axio-Observer Z1 inverted  Serial: 3851001242 Part Number: 431007-9902-000
    System ID: 1024979772

  • Camera adapter Model 60N-C, 1", 1x, Model: 426114

Light sources

  • Transmitted LED light
  • Colibri 7 R(G/Y)B-UV 423052-9730-000 Serial 5440000661

Condenser

  • Manual condenser Product number: TBD, Serial: TBD

  • Lens NA 0.35 WD 70 mm Part Number: 424241

  • Filter turret 6 positions manual

    1. H

    2. Ph0

    3. Ph1

    4. Ph2

    5. DIC

    6. DUC

Objectives

  • 2.5x/0.085 Air WD 8.8

  • Empty

  • 10x/0.25 Air Ph1 WD 6.5

  • Empty
  • 20x/0.5 Air Ph2 WD 2.0
  • 40x/0.95 Air WD 0.25

Stage

  • Motorized stage Marzhauser Sensotech, Part number 432903-9011-000, #14 07 132052; 90-76-200-0820
  • Remote control joystick
  • Inserts
    • Slide combo
    • 6-well plate
    • 35 mm dish
    • Multi-well plat

Filters

  1. DAPI Filter Set 49
  2. GFP Filter Set 13
  3. Rhodamine Filter Set 43
  4. DHE (dihydroethidium) 424931
  5. Cy5 Filter Set 50
  6. Multiband FS90 HE LED

Detector

  • Zeiss AxioCam MR R3 CCD Camera 1388 x 1040 pixels, 12-bit, 13 images/s at full resolution, detector size 8.9 mm x 6.7 mm. Model: r3.1 Part Number: 426509-9901-000. Serial: 1 22 12 5537

Workstation

  • Fujitsu Esprimo P920 E90+
  • Intel Core i5-4670 @ 3.4 GHz
  • RAM 32 GB DDR3 1600 MHz ECC (4 x 8 GB)
  • OS 500 GB SSD 550 MB/s
  • 2 TB HD Data Storage (2 x 1 TB spanned volume) 110 MB/s
  • Video Card AMD FirePro V4900 1 GB DDR5 dedicated memory
  • Monitor LG Flatron E2711 27' 1920 x 1080
  • Software Zen Blue 3.5 HASP YRMDZ 1798977001 1798977001

Incubation

  • Pecon stage top incubation

Consumables

  • CO2 Tank
  • N2 Tank
  • Oil
  • Lens Cleaner

Troubleshooting

I see a high background in fluorescence

The fluoresncece light source is a Colibri while the transmitted light is a LED. What happens is that the fluorescence illumination reflects and into the LED and give a high background. To solve this:

  • Tilt the transmitted light arm backward
    or
  • Stop the light from entering the transmitted LED by using a cardboard

FAQ

Can I use this microscope to look at cell in a dish?
  • Yes. 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
  • For long timelapse, be aware of photo-toxicity
Can I use this microscope to perform timelapse experiments?

Yes, but... This microscope has an incubation module to maintain temperature, humidity and gas. Yet it does not have a Definite focus which can maintain focus throughout time. Therefore, it is possible to loose the focus over long period.


Core Facilities CIB               Structural Biology    |    Cytometry    |    Microscopy    |    Histology