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Nikon Ti2-Oko microscope

Roger Gaudry Building, Room R-619
Advanced Microscope Tier 1 usage price
Instrument awarded to Dr Paradis-Bleau the Canadian Foundation for Innovation (CFI #34495) in 2015

 

Applications

  • Inverted microscope
  • Widefield imaging
    • Brightfield
    • Phase contrast
    • Polarized light, DIC
    • Fluorescence
  • Live imaging
  • Timelapse imaging
  • Incubation

Description

Light sources

  • LED lamp for transmitted light

  • Lumencor SpectraX for fluorescence

SourceFilterExcitation (nm)DluorophoresNominal power (mW)Measured Power at sample (mW)
Violet

395/25

[382-407]

DAPI, Hoechst

29559.4
Blue

440/20

[430-450]CFP25690.1
Cyan

470/24

[458-482]FITC, GFP19649.8
Teal

510/25

[497-522]YFP6215.8
Green/Yellow550/15

[542-557]

TRITC, Cy3

26069.9

Green/Yellow (Storage)

575/25[562-587]

mCherry

31078.2
Red

640/30

[625-655]

Cy5

23144.8

Objectives

  1. 4x/0.2 Air
  2. 20x/0.5 Air Ph1
  3. 20x/0.75 Air DIC
  4. 60x/1.4 Oil DIC
  5. 100x/1.45 Oil Ph3
  6. 100x/1.45 Oil DIC
PositionNameBrandFull nameIdentifierMagnificationNumerical apertureImmersionTypeWorking distance (mm)Transmittance
(% [nm])		ApplicationsCover glass thickness (mm)
14x/0.2
Air		Nikon4x/0.2
 Plan Apo Lambda

MRD00045

4x0.2AirPlan Apo Lambda20>80% [400-1000]BF, Fluo0.17
220x/0.5
Air Ph1		Nikon

20x/0.5 Ph1
 Plan Fluor

MRH1020120x0.5Air Plan Fluor2.1>80% [400-750]
Measured 72%		BF, Pol, PhC, Fluo0.17
320x/0.75
Air DIC		Nikon20x/0.75 DIC
Plan Apo Lambda
DIC N2		MRD00205

20x

0.75

Air

Plan Apo Lambda

1.0

>80% [400-950]
Measured 88%		BF, Pol, DIC, Fluo0.17
4

60x/1.4
Oil DIC

Nikon60x/1.4 Oil
 Plan Apo Lambda
DIC N2		MRD0160560x1.4Oil Plan Apo Lambda0.13>80% [475-725]
Measured 28%		BF, Pol, DIC, Fluo0.17
5
100x/1.45
Oil Ph3
Nikon100x/1.45 Oil
Plan Apo Lambda
Ph3		MRD31905100x1.45OilPlan Apo Lambda0.13>80% [475-750]
Measured 9%		BF, Pol, PhC, Fluo0.17
6100x/1.45
Oil DIC		Nikon100x/1.45 Oil
Plan Apo Lambda
DIC N2		MRD01905100x1.45OilPlan Apo Lambda0.13>80% [475-750]
Measured 11%		BF, Pol, DIC, Fluo0.17

Filters

  1. DAPI

  2. GFP/FITC (CFP)

  3. Cy5

  4. DAPI/GFP/Cy3/Cy5 (requires Cy3 filter in Lumencor SpectraX Green/Yellow position)

  5. DIC Analyzer

  6. CFP/YFP/mCherry (requires mCherry filter in Lumencor SpectraX Green/Yellow position)

PositionNameBrandIDExcitationDichroicEmissionComments
1DAPINikonDAPI-U HQ395/25x
[383-408]
303258		425LP

460/50m
[435-485]
307914

C-FL-C DAPI-U HQ
2GFPSemrockGFP-4050B-000

466/40x
[446-486]

495LP

525/50m
[500-550]		Nikon ID 96372
3Cy5Semrock

Cy5-5070A

618/50x
[5-6]		652LP698/70
[6-6]		Nikon ID 96376 M351081
4

DAPI/GFP/Cy3/Cy5

Semrock

C182279

None

409/493/573/652432/515/595/68177074160 Custom Quad C182279.
Excitation filters are in the Lumencor SpectraX light source
5

DIC Analyzer

NikonTi2-C-DICACLNot ApplicableNot ApplicableNot ApplicableUsed for DIC imaging
6

CFP\YFP\mCherry

SemrockC1997767None459/526/596

475/543/702

77074656 Excitation filters are in the Lumencor SpectraX light source

Detector

  • Hamamatsu ORCA Flash 4.0 V2 C11440-22CU

CameraHamamatsu ORCA Flash 4.0 V2

Sensor Type

CMOS

Sensor Category

Monochrome

Nb Pixels

4.2 M

Pixel Layout

2048 x 2048

Pixel size

6.5 um

Sensor size

13.312 mm x 13.312 mm 

Sensor diameter

 mm

Bit depth

16-bit
Speed at full resolution30 images/s

Max QE

83 %
Readout noise1.6 e⁻

Cooling

-10 C Air

Dark Current

0.06 e⁻/pixel/sec

Full well capacity

30 000 e-

Dynamic Range

1: 37 000

Interface

USB 3.0

Mount

C-mount

User Guide

  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. Turn on the microscope power bar (#2) on the desk between the microscope and the computer
  4. if incubation is required, turn on Okolab incubation module (#3A), the Lauda water bath (#3B) and open the CO₂ (#3C) and N2 (#3D) cylinders near the door

    Make sure the humidifier and water bath are properly filled with distilled water.

  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 before starting the software
  6. Start NIS-Elements

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. If you are not sure, ask for support.

  1. If NIS-Elements 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. Open NIS Settings Utility
  4. Click on the Import tab
  5. Click on Browse
  6. Navigate to your C:\Users\Public\Documents
  7. Select the file Nikon_Ti2-Oko_NIS Settings.bin
  8. Click Select
  9. Select all items
  10. Click Import
  11. Click OK
  12. Close the NIS Settings Utility
  13. You can now reopen NIS-Elements

During this procedure, you will:

  • Set the microscope in a safe configuration

  • Load your sample

  • Find and adjust the focus

Once completed, your sample will be ready for acquisition.

  1. On the microscope, gently push the transmitted light arm backward.

  2. In NIS-Elements, click Escape to move the objectives to a safe position.

  3. Click on the lowest magnification objective.

    The lowest magnification is the safest to use due to its long working distance: the sample will appear in focus well before the objective lens gets close to it. It is recommended to always perform the initial focusing with the lowest magnification objective. Since the objectives are parfocal, focusing with these objectives will make it easier to locate the sample when switching to higher magnification objectives.

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

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

  5. If necessary, use the joystick to move the stage so the sample is centered under the objective.

  6. Gently return the transmitted light arm to the vertical position.

  7. In NIS-Elements, click Escape again to return the objectives to their normal position.

  8. Click on the desired optical configuration (BF, DAPI, GFP, etc.).

  9. Click Live (») to activate the light and display the camera image.

  10. Adjust the light intensity and exposure time to obtain a well-exposed image.

  11. Adjust the focus until the image is perfectly sharp.

  12. Click Stop to stop the Live or on Off to turn off the light

The focus is around Z = 2100 µm. The Z-position value is displayed on the microscope remote control screen.

First perform the initial focusing with the safest objective before selecting a higher-magnification objective.

After completing the initial focus:

  1. In NIS-Elements, click on the desired objective (20x PhC or 20x DIC)

    The 20x DIC objective is the best air objective because it has the highest numerical aperture (0.75).

  2. Click on the desired optical configuration (BF, DAPI, GFP, etc.).

  3. Click Live (») to activate the illumination and display the camera image on the screen.

  4. Adjust the light intensity and exposure time to obtain a properly exposed image.

  5. Adjust the focus using the precision knob until the image is perfectly sharp.

  6. Click Stop to stop the Live mode or click on the optical configuration Off to turn off the illumination.

  7. Click Escape to move the objectives to a safe position.

  8. On the microscope, you can remove the test slide and install your sample.

  9. In NIS-Elements, click Escape once again to return the objectives to their normal position.

Your sample is now ready for acquisition!

After completing the initial focusing:

  1. In NIS-Elements, click Escape to move the objectives to a safe position.

  2. Click on the 63x, 100x DIC, or 100x PhC objective to select the desired lens.

    The 100x DIC objective is the best oil immersion lens because it has the highest numerical aperture (1.45).

  1. Place a single drop of oil on the objective.

  2. Click Escape again to return the objectives to their normal position.

  3. Click on the desired optical configuration (BF, DAPI, GFP, etc.).

  4. Click Live (») to activate the illumination and display the camera image on the screen.

  5. Adjust the light intensity and exposure time to obtain a properly exposed image.

  6. Adjust the focus using the precision knob until the image is perfectly sharp.

  7. Click Stop to stop the Live mode or click on the optical configuration Off to turn off the illumination.

Your sample is now 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 NIS-Elements
  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 lowest magnification objective and press escape to place the objectives in a safe position

  6. If used, turn off the Okolab incubation module (#3A), Lauda water bath (#3B) and close the CO2 (#3C) and N2 (#3D) cylinders

  7. Wait until the SpectaX fan is off and then turn off the microscope power bar (#2)

  8. Turn off the computer
  9. 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

Log

  • Condenser DIC prism is DIC N1 while objective requires N2
  • Check why at 4x Condenser gives a black shadow on the righ side of the image
  • NIS Objective Z offset
  • Nitrogen Tank replacement Praxair 1066 106723504
  • CO2 Tank replacement Praxair 1013 106722901
  • Migrated to Windows 11
  • Fixed Stage insert bent
  • Cleaning all objective and filter cubes
  • Added XY Offset
  • Fixed incubator tubing misplaced
  • Added better attachment for tubing on the incubation chamber
  • Checked incubation chamber for obstruction
  • Fixed incubator tubing misplaced
  • Added better attachment for tubing on the incubation chamber
  • Checked incubation chamber for obstruction
  • Fixed condenser holder loose
  • 20x/0.5 Ph1 dirty or damaged likely dirty with varnished
  • Added network cable between Acquisition and Processing workstations
  • Added shortcuts in Desktop\Documentation
  • Clean 60x objective (nail polish on it !)
  • Fix damaged stage insert
  • Stage insert tilt adjustment
  • Objective XY offset adjustments
  • Installed new objective 4x/0.2 Plan Apo Lambda
  • Updated configuration file
  • Computer maintenance
  • Imaging test slides
  • Re-install DCAM API drivers v22.2.6391
  • Okolab incubation complete maintenance
    • Disassemble Okolab stage insert
    • Disconnect the pipes
    • Disconnect the Lauda water-bath
    • Clean everything with 10% acetic acid (excepted the plastic pipes and washers)
    • Rinse with regular tap water 2 times
    • Rinse with distilled water once
    • Dry, reassemble and reconnect
    • Refill with the Lauda water-bath with 3L of milliQ water
    • Set the Okolab temperature Control Mode to Chamber
    • Set the Okolab temperature to 65°C
    • Let run for 1h. This will sterilize the water.
    • Check for any leakage
    • Set the Okolab temperature Control Mode back to Sample
    • Set the Okolab temperature back to 37°C
  • Cleaning objectives
  • Control for incubation chamber liquid intrusion
  • CO2 Calibration for Okolab: Offset of 1%
  • Control for illumination quality
  • See 
  • Liquid light guide adjustment
  • 20x/0.75 DIC objective was dirty and has been cleaned
  • Printed and displayed a Memo about available air and oil objectives
  • Adjustment of camera angle
  • Objective calibration
  • Objective XY offset
  • Updated NIS settings
  • Added light path schematics to Wiki
  • Okolab display a NAN message for the free thermal sensor

  • The probe is made of two wires that need to be in contact to measure the temperature properly

    • Turn off the Okolab module
    • Disconnect the free sensor probe
    • Cut out the damaged part
    • Carefully expose the two wires
    • Connect the two wires together
  • Added label on gas bottles
  • Added line mark on humidifier
  • 512 GB SSD installed for OS
  • Windows 10 Installation
  • BIOS updated to v2.47

Technical Datasheet

Stand

  • Nikon Ti2-E inverted  Serial 540156 System 170110-Sys-006287

Light sources

  • Transmitted LED light
    • ND32 filter
    • IR filter
    • Manual Polarizer
  • Lumencor SpectraX 6-NII-SE Serial 9409

Condenser

  • Motorized condenser Ti2-C-TC-E Serial 519097

  • Lens LWD NA 0.52

  • Filter turret 7 motorized positions

    1. Empty

    2. Empty

    3. Ph1

    4. Ph3

    5. Shutter

    6. Empty

    7. DIC N1

Objectives

  1. 4x/0.2 Air MRD00045
  2. 20x/0.5 Air Ph1 MRH10201
  3. 20x/0.75 Air DIC MRD00205
  4. 60x/1.4 Oil DIC MRD01605
  5. 100x/1.45 Oil Ph3 MRD31905
  6. 100x/1.45 Oil DIC MRD01905

Stage

  • Motorized stage Ti2 SHU compatible Serial 127808
  • Remote control joystick Ti2-S-JS Serial 127976
  • Inserts
    • Multi-well plate Ti2-S-HW with tilt adjustment (no incubation)
    • Combo slide 3 cm dish  Ti2-S-HU with tilt adjustment (no incubation)
    • Okolab H101-CellASIC Frame with perfusion ports
      • 1 x 35mm petri dish 1x35-M + cover
      • 2 Chamber Slide 2xGS-M+ cover
      • 1 Multi-well for oil objectives MW-OIL
      • 6-well plate 6MW+ cover
      • 1 CellASIC + cover

Filters

  1. DAPI Cube Ex 383-408 DAPI-U DM 425 BA 435-485
  2. GFP Cube Semrock 96372 M349727 17
  3. Cy5 Cube Semrock 96376 M351081 8
  4. 77074160 Custom Quad C182279 Polychroic and quad bandpass emitter for use with the following single bandpass filters: ET395/25x, ET470/24x, ET550/15x, ET640/30x
  5. DIC Analyser Ti2-C-DICACL
  6. C197767 7707\4656 CFP\YFP\mCherry XT

Detector

  • Hamamatsu ORCA Flash V2 C11440-22CU Serial 101081

Workstation

  • HP Z440 Workstation
  • I155431-CIB
  • Intel Xeon E5-1620 v4 @ 3.5GHz
  • RAM 32 GB DDR4-2400 1200 MHz ECC (4 x 8 GB)
  • OS 500 GB NVMe via PCIe to M2 Adapter 2 500 MB/s
  • 4 TB HD Data Storage (2 x 2 TB spanned volume) 130 MB/s
  • Video Card nVidia GTX 1080 8GB GDDR5 dedicated memory
  • Monitor HP Z24i display 24' 1920x1200
  • Software NIS-Elements AR v5.02

Incubation

  • Okolab BoldLine Temperature unit Serial 284-1058 H101 T Unit BL
  • Okolab BoldLine CO2/O2 Unit  0-10/1-18 Serial 088-1102
  • Okolab OkoTouch Serial 118-224
  • Lauda water-bath Model Eco RE415 S LCK 4910 Serial LCK-4910-16-0006 Okolab 1322-1006 2017-05-30

Anti-vibration Table

  • TMC #63463542-03 Serial 205235

Consumables

  • CO2 Tank
  • N2 Tank
  • Liquid Light Guide

Manuals

Troubleshooting

This can happen when the liquid running through the chamber is not flowing properly.

  • Pause your experiment
  • Turn off the Okolab environment controller 3A and 3B
  • Disconnect the blue end of the connection pipe (at the junction with the spring shape objective warmer)
  • Place the open end into a dish to collect liquid
  • Turn the Lauda water bath back ON (3B)
  • The liquid should flow quite rapidly, if not proceed as follow
    • Turn OFF the Lauda water bath (3B)
    • Take the 20 mL syringe located inside the drawer labelled Tubing
    • Connect it to the open end of the blue pipe
    • Use the syringe to blow pressurized air into the pipes to clear it out
    • Remove the syringe (and store it back into the Tubing drawer)
    • Test if the liquid is now flowing properly by turning the Lauda water bath back ON (3B)
      • If not repeat the procedure
      • If so, turn OFF the Lauda water-bath (3B)
      • Reconnect the blue and green pipes together
      • Turn the Okolab environment controller 3A and 3B back ON

Be careful not to touch your sample when performing these actions as it may displace your current acquisition

How the Okolab chamber works

  1. The liquid from the water bath enters the lead first via the red pipe

  2. Then it goes through the lead circuitry to keep it warm
  3. It exists via the unlabelled tube and enters the incubation chamber main body
  4. It goes through the circuitry of the main body and exits via the green labelled tube

One must maintain a decent amount of liquid in the Lauda water bath to avoid bubble formation in the circuitry.
Because the circuitry inside the top lead and the incubation chamber main body is thin it can get clogged. The procedure above can solve this issue.

This happens when the mixed-gas humidifier is overfilled. Bubbles created by the gas going through the humidifier can bring liquid into the gas feed line.

  • Turn off the Okolab module and the water bath
  • Remove your sample and store it properly
  • Dry the incubation chamber with a clean tissue
  • Carefully remove the cap of the humidifier glass bottle

Pay extra care when manipulating the humidifier bottle as it is made of glass and is very fragile

  • Remove distilled water from the humidifier

Humidifier shouldn't be more than 2/3rd filled

  • Close the humidifier by replacing the cap
  • Turn on the Okolab module and the water bath

This can happen during long experiments. The high humidity of the gas mixture condensates in the pipe between the humidifier and the incubation chamber.

  • Pause your experiment
  • Disconnect both ends of the yellow tube from the humidifier
  • Drain the tube from any liquid
  • Reconnect the yellow tube to the humidifier
  • You can use a syringe or gas pressure to blow out any liquid from the incubation chamber cover

Be careful not to touch your sample when performing this action

  • Wipe any liquid with a tissue
  • Reconnect the yellow tube to the incubation chamber 

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

You may use a trick to warm-up the incubation chamber faster. Using this method. you should reach a stable temperature within 30 minutes.

Do NOT proceed with your sample but with a blank control (dish with distilled water for example)

  • Place your blank control in the incubation chamber
  • Immerse the tip of the sample temperature probe in the blank
  • Set the Okolab temperature Control Mode to Chamber (Settings>Temperature>Control Mode>Chamber>Save)
  • Set the Okolab temperature to 50°C (Home>Temperature>50°C>Set)
  • Check the temperature of the sample (Menu Magnifier>Sample Temperature). It should take between 10 to 15 minutes for the blank to reach 30°C. 

The Lauda water bath is faster to warm up water than to cool it down. Make sure to anticipate and not pass beyond the desired temperature.
If your desired temperature is 37°C, you can stop the procedure when the blank has reached 30°C.

When the blank has reach the desired temperature

  • Set the Okolab temperature back to 37°C (Home>Temperature>37°C>Set)
  • Set the Okolab temperature Control Mode to Sample (Settings>Temperature>Control Mode>Sample>Save)
  • Wait 10 to 20 minutes until the temperature stabilizes
  • Then you can safely replace the blank with your sample

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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"