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Zeiss Axio-Observer Z1 Colibri inverted microscope

Desmarais Building, Room 2234
Simple Microscope usage price

Instrument awarded to Dr. Audrey Claing and Dr. 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 (385/469/555-590/631) for fluorescence

      Emission peak (nm)

      		Power (mW)
      385/30150
      469/38110
      555/3031
      631/3350

      Information Zeiss Colibri 7


  • Objectives

    1. 2.5x/0.075 Air

    2. 10x/0.25 Air Ph1
    3. 20x/0.5 Air Ph2

    4. 40x/0.75 Air Ph2

    5. 63x/0.75 Air Ph2 Long Distance
    6. 63x/1.4 Oil
      PositionNameBrandFull nameIDMagnificationNumerical ApertureImmersionTypeWorking distance (mm)Transmittance
      (% [nm])      		TechniqueCover glass thickness (mm)
      12.5x/0.075 AirZeiss2.5x/0.075
      EC Plan-Neofluar
      M27      		420320-9901-0002.5x0.075AirPlan Neofluar9.5>80% [400-840]BF, Fluo0.17
      210x/0.25 AirZeiss10x/0.25 Ph1
      N-Achroplan
      M27       		420941-9911-00010x0.25AirN-AchroPlan

      6.5

      		Not availableBF, PhC, Fluo0.17
      320x/0.5 AirZeiss20x/0.50 Ph2
      EC Plan-Neofluar
      M27

      420351-9910-000

      		20x0.50AirPlan Neofluar2.0Not AvailableBF, PhC, Fluo0.17
      4

      40x/0.75

      		Zeiss40x/0.75 Ph2
      EC Plan-Neofluar
      M27

      420361-9910-000

      40x

      		0.75

      Air

      		Plan Neofluar0.71Not AvailableBF, PhC, Fluo

      0.17

      563x/0.75Zeiss

      63x/0.75 Corr Ph2
      LD Plan-Neofluar
      M27

      		421381-9970-000

      63x

      0.75

      		AirLD Plan-Neofluar1.7 at cover glass 0.75 Not AvailableBF, PhC, Fluo

      0 - 1.5

      663x/1.4Zeiss63x/1.4 DIC
       Plan-Apochromat Oil M27      		420782-9900-00063x1.4OilPlan Apochromat0.19>80% [400-700]BF, 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]
      3DsRed
      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      		Zeiss489090-9110-000


      		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

  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. Log in Windows using your UdeM 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.

  6. Start the Zen 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.

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

  1. If open, close Zen and wait for it to close completely (up to 30 seconds)
  2. On the Desktop open the Documentation folder
  3. Double-click Zen Settings for Axio-Osberver Z1-Colibri
  4. A script will run and a black window will appear briefly
  5. Click OK to close the message Settings for Zen have been imported successfully.
  6. You can then reopen Zen 

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.

On the microscope touch screen:

  1. Press Home>Load Position to lower the objectives 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>Control>Objectives>2.5x to select the 2.5x 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 = 1.1 mm). The Z value can be found on the microscope touch screen Home>Z-Position

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 2.5x to select the 2.5x lens

    The 2.5x objective is the safest because it has the longest working distance (9 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 objectives 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 Zen
  2. In the Locate tab, select BF or the desired fluorescence (DAPI, GFP, Rhodamine, 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 =  1.1mm). 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

Important

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

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

  1. Press Home>Microscope>Control>Objectives
  2. Press 20x or 40x or 63x to select the desired lens

    The 40x objective is the best Air objective because it has the largest numerical aperture (0.75).


  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!

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

  1. Press Home>Microscope>Control>Objectives

  2. Press 63x Oil. The microscope will automatically lower the objective so that the sample is accessible

  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:

  1. In the Locate tab, select BF or the desired fluorescence (DAPI, GFP, Rhodamine, 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!
  • 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.

  1. Save your data
  2. Close Zen
  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 2.5x 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.


  • Microscope Firmware update to add Colibri to the touchscreen
  • Zen 3.5 HotFix 10
  • Computer replacement
  • Added Colibri
  • Added startup procedure
  • Parafocality and paracentrality
  • Added complete description
  • 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

  1. 2.5x/0.075 Air 420320-9901-000

  2. 10x/0.25 Air Ph1 420941-9911-000

  3. 20x/0.5 Air Ph2 420351-9910-000

  4. 40x/0.75 Air Ph2 420361-9910-000

  5. 63x/0.75 Air Ph2 Long Distance 421381-9970-000

  6. 63x/1.4 Oil 420782-9900-000

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 488049-9901
  2. GFP Filter Set 13 488013-0000
  3. Rhodamine Filter Set 43 000000-1114-101
  4. DHE (dihydroethidium) 424931
  5. Cy5 Filter Set 50 488050-9901
  6. Multiband FS90 HE LED 489090-9110-000

Detector

  • Zeiss AxioCam MR R3 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

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

  • 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
  • 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.
Zeiss Z1 Colibri


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