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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) in 2014

  • Transmitted light
  • Brightfield
  • Phase contrast
  • Fluorescence
  • Timelapse
  • Incubation
  • Deconvolution
  • Extended Focus

Zeiss Z1 Colibri

Light sources

  • LED lamp for transmitted light

  • Colibri 7 R[G/Y]B-UV (385/469/555-590/631) for fluorescence

Emission peak (nm)

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

Zeiss Colibri 7 Datasheet

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 Air

Zeiss40x/0.75 Ph2
EC Plan-Neofluar
M27

420361-9910-000

40x

0.75

Air

Plan Neofluar0.71Not AvailableBF, PhC, Fluo

0.17

563x/0.75 Air
Zeiss

63x/0.75 Corr Ph2
LD Plan-Neofluar
M27

421381-9970-000

63x

0.75

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

0 - 1.5

663x/1.4 OilZeiss63x/1.4 DIC
 Plan-Apochromat Oil M27		420782-9900-00063x1.4OilPlan Apochromat0.19>80% [400-700]BF, Fluo0.17

Filter

  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

Detectors

  • Zeiss AxioCam MRm
CameraZeiss AxioCam MRm

Sensor Type

CCD

Sensor Category

Monochrome

Nb Pixels

1.4 M

Pixel Layout

1388 x 1040

Pixel size

 6.45 um

Sensor size

8.9 mm x 6.7 mm

Sensor diameter

11 mm

Bit depth

12-bit
Speed at full resolution13 images/s

Max QE

 55 %
Readout noise 8 e⁻

Cooling

Pelletier

Dark Current

0.7 e⁻/pixel/sec

Full well capacity

17 000 e-

Dynamic Range

1:2000

Interface

FireWire (IEEE 1394a)

Mount

C-mount


Zeiss_AxioCam MRm_Datasheet.pdf

  • PCO Edge 5.5
CameraPCO Edge 5.5

Sensor Type

sCMOS

Sensor Category

Monochrome

Nb Pixels

5.5 M

Pixel Layout

2560 x 2160

Pixel size

 6.5 um

Sensor size

16.6 mm x  14.0 mm

Sensor diameter

21.8 mm

Bit depth

16-bit
Speed at full resolution100 images/s

Max QE

 60 % at 600 nm
Reading noise 1.0 e⁻

Cooling

Forced air +7C

Dark Current

0.6 e⁻/pixel/sec

Full well capacity

30 000 e-

Dynamic Range

1:30000

Interface

Dual Camera Link PCIe

Mount

C-mount

PCO_Edge 5.5_Datasheet.pdf

  1. If not already done, turn on the computer (#1) and log in to Windows using your UdeM credentials
  2. Remove the dust cover from the microscope
  3. If incubation is required, turn on the incubation power bar (#2) on the desk near the computer and open the CO₂ cylinder (#2B) near the sink

  4. Turn on the microscope power bar (#3) on the desk near the computer

  5. When using the instrument for the first time, it is necessary to import the microscope configuration into the software. See the First Use section below.
  6. Start Zen

When using the instrument for the first time, it is necessary to import the microscope configuration into the software. This procedure is usually carried out during the training session. However, it can also be used to reset the software if it does not display correctly, for example. Running this procedure will erase all your experiment protocols and reset the software to its original settings (ask for support if you are not sure).

  1. If Zen is open, close it and wait until it has completely shut down (this may take up to 30 seconds)

  2. On the Desktop, open the Softwares folder

  3. Double-click Zen Settings for Axio-Observer Z1-Colibri

  4. A script will run and a black window will appear briefly

  5. When the message Settings for Zen have been imported successfully appears, click OK to close it

  6. You can now open Zen

During this procedure, you will:

  • Set the microscope to a safe configuration
  • Perform a calibration
  • Load your sample
  • Find and adjust the focus

Once completed, your sample will be ready for acquisition.

This step is required to calibrate the microscope in XY and Z. Performing this calibration will significantly reduce the time needed to locate and focus on your sample.

  • If not already done, select the lowest-magnification objective. On the microscope touch screen Home > Microscope > Control > Objectives > 2.5x
  • In Zen, once it has started a calibration dialog should appear. Simply click Calibrate Now.
    The microscope will lower the objectives, perform an XY calibration first, followed by a Z calibration, and then return to its original position.

Once calibrated, the focus is typically found at Z = 1.1 mm for a microscope slide.
The Z position can be viewed on the microscope touch screen under Home > Z-Position, as well as in Zen within the Focus tab, located on the right side of the screen.

The system may already be calibrated. This can occur if a previous user calibrated the system and left it on.

In Zen, within the Focus tab, located on the right side of the screen:

  1. Click Load to lower the objective

  2. The Z position is indicated below Current
  3. If Z position value is less than 100 um the system is calibrated

On the microscope touch screen:

  1. Lower the objective by pressing Home > Load Position

  2. Press Set Work Position to store this position

  3. If necessary, slightly adjust the focus upward to clear the Lower Z Limit Reached message displayed on the touch screen

  4. The Z position value is indicated
  5. If the value is less than 0.1 mm then the system is calibrated

In Zen, within the Stage tab, located on the right side of the screen:

  1. If not already done, check the Show All option

  2. At the bottom of the tab click Calibrate

  3. A warning message will show up click Continue

  4. The microscope will lower the objectives and perform a XY stage calibration and then return to its original position.

In Zen, within the Focus tab, located on the right side of the screen:

  1. If not already done, check the Show All option

  2. At the bottom of the tab click Calibrate

  3. A warning message will show up click Continue

  4. The microscope will then perform a Z calibration and then return to its original position

XY and Z calibrations are now complete

On the microscope touch screen:

  1. Navigate to Home > Microscope > XYZ > Position > Z-Position > Set Zero > Auto to perform focus calibration

  2. Press OK to start the calibration procedure

  3. Wait a few seconds for the calibration to complete

  4. Lower the objective by pressing Home > Load Position

  5. Press Set Work Position to store this position

  6. If necessary, slightly adjust the focus upward to clear the Lower Z Limit Reached message displayed on the touch screen

  7. Navigate to Home > Microscope > XYZ > Position > XY-Position > Set Zero > Auto to perform a stage calibration

  8. Press OK to start the calibration procedure

  9. Wait a few seconds for the calibration to complete

XY and Z calibrations are now complete

In Zen:

  1. In the menu bar, navigate to Tools > Options

  2. Select Startup/Shutdown

  3. Under Stage/Focus Calibration, ensure Request Stage/Focus Calibration on Startup is checked

  4. Click OK to close the Options dialog

Important

Ensure that the calibration has been completed beforehand. Calibration will significantly reduce the time required to locate and focus on your sample.

On the microscope touch screen:

  1. If not already done, select the lowest-magnification objective. On the microscope touch screen Home > Microscope > Control > Objectives > 2.5x

The 2.5× and 10× objectives are the safest to use due to their long working distance (>6 mm). The sample will appear in sharp focus well before the objective approaches it. It is recommended to always focus using the safest objectives first. Since the objectives are parafocal, focusing with the safest objectives will facilitate locating the sample when switching to higher-magnification objectives.

  1. Lower the objective by pressing Home > Load Position

  2. Press Set Work Position to store this position

  3. If necessary, slightly adjust the focus upward to clear the Lower Z Limit Reached message displayed on the touch screen

  4. Place the test slide on the microscope stage with the coverslip facing the objective

    Important

    Using a test slide will significantly reduce the time required to set up the instrument.

  5. If necessary, adjust the stage to ensure that the sample is centered under the objective

In Zen:

  1. In the Locate tab, select BF or the desired fluorescence (Fluo, DAPI, GFP, Cy3, Cy5) to activate the configuration
  2. Adjust the focus with the main dial while looking through the eyepieces until the image is perfectly sharp

    Once calibrated, the focus is typically found at Z = 1.1 mm for a microscope slide.
    The Z position can be viewed on the microscope touch screen under Home > Z-Position, as well as in Zen within the Focus tab, located on the right side of the screen.


  3. In the Locate tab, select Off to turn off the illumination

Important

Perform the initial focus using the safest objective before switching to higher-magnification objectives.

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

  1. Press Home>Microscope>Control>Objectives
  2. Press 10x, 20x, 40x or 63x (0.75) to select the desired objective
The 40x objective is the best Air objective because it has the largest Numerical aperture (0.75) together with a wider field of view (40x).

In Zen:

  1. In the Locate tab, select BF or the desired fluorescence (Fluo, DAPI, GFP, Cy3, Cy5) 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 off the illumination

Your sample is ready for acquisition!

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

  1. Press Home>Microscope>Turret>Objectives

  2. Press 63x Oil (1.4) to select the desired objective. The microscope will automatically lower the stage so that the sample becomes accessible.

  3. Place a single drop of oil on the objective
  4. Press Done. The microscope will automatically return the objective to its original position

In Zen :

  1. In the Locate tab, select BF or the desired fluorescence (Fluo, DAPI, GFP, Cy3, Cy5) 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 off the illumination

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, clean oil objectives with lens cleaner and paper
  5. Select the 2.5x objective and press load position to place the objectives in a safe position

  6. If used, turn off the incubation module power strip (#2A) and close the CO2 cylinder (#2B)

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

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

Reminder

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

  • AxioCam replacement with PCO Edge 5.5
  • Updated wiki with complete specifications
  • 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

  • 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 Scan IM 130x100-2mm 90-24-550-0000 Serial 16053038
    Stage Controller 90-76-024-1803 Serial 14 04 1 2040 SMC 2009 432929-9011-000
  • Remote control joystick 2-Axis 90-76-200-0820 Zeiss Article 432903-9011-000 Serial 1615142059
  • Inserts
    • Slide combo
    • Multi-well plate

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 MRm Model: r3.1 Part Number: 426509-9901-000. Serial: 1 22 12 5537
  • PCO Edge 5.5 Model pco.API.Air.BX Serial 6000001404

Workstation

  • Fujitsu Esprimo P920 E90+ Model MI5W Serial YLPS036541
  • Intel Core i5-4670 @ 3.4 GHz
  • RAM 24 GB DDR3 800 MHz ECC (3 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 and LG W2442 1920 x1080
  • Software Zen Blue 3.5 SN=1121159628-524292 HASP=1798977001 Extended Focus, Measurements, Multi-Channel, Panorama, Software autofocus, Time Series, Z-stack 

Incubation

  • Pecon stage top incubation

Consumables

  • CO2 Tank
  • N2 Tank
  • Oil
  • Lens Cleaner

Troubleshooting

This happens in the following conditions at

  • At 2.5x using the DAPI or GFP channel
  • At 10x using the GFP channel
  • At 20x using the GFP channel

 To solve this you can use any of the following option:

  • Tilt the transmitted light arm backward
  • Close the manual shutter between the condenser and the transmitted light
  • Use a higher magnification objective

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. You can always do a software autofocus at different time but be aware of photo-toxicity if you are using fluorescence.

Extended Focus is a method to flatten a Z-stack by keeping only the relevant information.

In Zen:

  • Select the Processing Tab
  • Select the Extended Depth of Focus Method
  • Click Apply
  • Wait until the processing is finished
  • Save the processed image

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Content published under CC BY-SA 4.0. Sharing allowed under the same license with attribution: "Original content by l'Institut Courtois d'innovation biomédicale, used under CC BY-SA 4.0"

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