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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
  • Deconvolution

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 

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 tank 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, 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

  • 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. To verify whether the system is calibrated, simply…


O

the calibration popup dialog did not show up you at Zen opening, you can do it manually:

Select the Stage window (on the right of Zen window) and click Calibrate

A warning message will show up click Continue

The microscope will lower the objectives perform a XY calibration

Then in the Focus Window on the right of Zen window) and click Calibrate

A warning message will show up click Continue

The microscope will lower the objectives perform a Z calibration

 Done.

nce calibrated, the focus can be found at Z = 1.1 mm for a microscope slide). The Z value can be found on the microscope touch screen Home>Z-Position

Once calibrated, the focus can be found at Z = 1.7 mm). The Z value can be found on the microscope touch screen Home>Z-Position

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

  10. Calibration is now complete


On the microscope touch screen:

  1. If not already done, Press Home>Load Position to lower the objectives to the 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. If not already done, press Home>Microscope>Control>Objectives>5x to select the 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 calibration procedure
  8. Wait a few seconds for the calibration to be completed
  9. Press Home>Microscope>XYZ>Position>XY-Position>Set zero>Auto to perform a stage calibration
  10. Press OK to start the calibration procedure
  11. Wait a few seconds for the calibration to be completed

Once calibrated, the focus can be found at Z = 1.7 mm). The Z value can be found on the microscope touch screen Home>Z-Position



I


 


If

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

On the microscope touch screen:

  1. If not already done, press Home>Microscope>Turret>Objectives>5x to select the 5x objective

    The 5x objective is the safest because it has the longest working distance (12mm). 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 parafocal, focusing with the safest objective will then allow you to easily find your sample with another objective. The 10x objective is also safe because its working distance is 6.5 mm.

  2. If not done already, press Home>Load Position to lower the objective to the lowest position and 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.

  5. 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, mPlum) 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 = 1.7 mm). 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

First focus with the safest objective 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, press 10x, 20x or 40x to select the desired objective
    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) but has a smaller field of view.
    The 20x/0.8 objective offers the best compromise between Resolution and Field of View

    There are two (2) 40x objectives, make sure you select the Air 40x


In Zen :

  1. In the Locate tab, select BF or the desired fluorescence (DAPI, GFP, mPlum) 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, 40x Oil to select the desired objective. The microscope will automatically lower the stage so that the sample is accessible.

    The 40x and 63x oil objectives provide the same spatial resolution because they have the same numerical aperture (1.4).
    The 40x oil objective offers a larger field of view and transmits light slightly better beyond 700nm.
    The 63x oil objective transmits light slightly better in the visible spectrum (440-710 nm) and has a better Strehl ratio (90%). It is particularly suited for super-resolution imaging, but its field of view is smaller.

    There are two (2) 40x objectives, make sure you select the 40x Oil


  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 (DAPI, GFP, mPlum) 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!

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

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

    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


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
  • 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.


Available manuals



  • 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 SN=1121159628-524292 HASP=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.