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Nikon Ti2-E fully motorized inverted microscope

Roger Gaudry R-619

  • Applications

    • Brightfield

    • Phase contrast

    • Polarized light, DIC

    • Fluorescence

    • Live imaging
    • Long timelapse imaging

  • Light sources

    • LED lamp for transmitted light

    • Lumencor SpectraX for fluorescence 

Light sourceFilter IDFilter typeExcitation wavelengths (nm)Compatible fluorophoresPower (mW)
Violet

395/25

Bandpass[382-407]

DAPI, Hoechst

295
Blue

440/20

Bandpass[430-450]CFP256
Cyan

470/24

Bandpass[458-482]FITC, GFP196
Teal

510/25

Bandpass[497-522]YFP62
Green/Yellow550/15Bandpass

[542-557]

TRITC, Cy3

260

Green/Yellow (Storage)

575/25Bandpass[562-587]

mCherry

310
Red

640/30

Bandpass[625-655]

Cy5

231

  • Objectives

    • 20x/0.5 Air Ph1
    • 60x/1.4 Oil DIC WD 0.13
    • 100x/1.45 Oil Ph3 WD 0.13
    • 100x/1.45 Oil DIC WD 0.13
    • Empty
    • 20x/0.75 Air DIC 
PositionNameBrandFull nameProduct IDMagnificationNumerical ApertureImmersionTypeWorking distance (mm)Transmittance
(% [nm])		TechniqueCover glass thickness (mm)
120x/0.5 Air Ph1Nikon

20x/0.5 Air Plan Fluor Ph1

MRH1020120x0.5AirPlan Fluor2.1>80% [400-750]BF, Pol, PhC, Fluo0.17
2

60x/1.4 Oil DIC

Nikon60x/1.4 Oil Plan Apo Lambda DIC N2MRD0160560x

1.4

Oil
Plan Apo Lambda0.13>80% [475-725]BF, Pol, DIC, Fluo0.17
3

100x/1.45 Oil Ph3

Nikon100x/1.45 Oil Plan Apo Lambda Ph3MRD31905

100x

1.45
Oil
Plan Apo Lambda0.13>80% [475-750]BF, Pol, PhC, Fluo0.17
4100x/1.45 Oil DICNikon100x/1.45 Oil Plan Apo Lambda DIC N2MRD01905

100x

1.45

Oil
Plan Apo Lambda0.13>80% [475-750]BF, Pol, DIC, Fluo0.17
5Empty










620x/0.75 Air DICNikon20x/0.75 Air Plan Apo Lambda DIC N2MRD0020520x0.75AirPlan Apo Lambda

1.0

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

  • Filter cubes

    1. DAPI

    2. GFP/FITC (CFP)

    3. Cy5

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

    5. DIC Analyzer
    6. CFP/YFP/mCherry (requires mCherry filter in Lumencor Green/Yellow position)
PositionCube nameBrandIDExcitation FilterDichroic mirrorEmission FilterComments
1DAPINikonDAPI-U HQ395/25x [383-408]425LP460/50m [435-485]C-FL-C DAPI-U HQ
2GFPSemrockGFP-4050B-000

466/40x [446-486]

495LP

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

Cy5-5070A

617/55x [590-645]652LP697/77m [659-736]Nikon ID 96376
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

Excitation filters are in the Lumencor SpectraX light source

  • Detector

    • Hamamatsu ORCA Flash V2 C11440-22CU CMOS Monochrome Camera 2048 x 2048 pixels, 16-bit, 30fps at full frame

  1. Turn on the computer
  2. Turn on the microscope power bar

  3. If incubation is required, turn on the Okolab incubation module (3A and 3B) and open the CO2 (3C) and N2 (3D) tanks

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

  4. Log in Windows using your UdM credentials
  5. Start-up NIS-Elements

The first time you log in the computer, you need to import the microscope settings. To do this follow the instructions Setting-up NIS-Elements

  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. Get your samples from the microscope
  5. Clean the oil objectives with lens cleaner and lens paper
  6. If incubation was used, turn off the incubation module (3A and 3B) and close the CO2 (3C) and N2 (3D) tanks
  7. Turn off the microscope power bar (2)
  8. Cover the microscope

Important Reminders

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

This process is required the first time you are using the instrument. You will usually do it during the training session. It can also be performed if something is not working properly right or if you want to refresh the software interface to the original settings.

This process will delete all experiment protocols and restore the parameters for the microscope.

  1. Close NIS-Elements
  2. Wait until the complete closure of NIS-Elements
  3. Open the folder Desktop\Logiciels
  4. Open the software NIS Settings Utility
  5. Click on the Import tab
  6. Click on Browse
  7. Navigate to your desktop
  8. Select the file Nikon-Ti2 Settings.bin
  9. Click Select
  10. Select all items
  11. Click Import
  12. Click OK
  13. Close the NIS Settings
  14. Open NIS-Elements

Light path

Download the schematics to follow Transmitted light (Bright field, Phase Contrast, Polarized light, DIC) and Reflected light (fluorescence) paths.

LightPath.pdf

  • Bring long Rj45 cable to connect Acquisition and Processing workstations
  • Check condenser filters if labels match
  • Check why 20x/0.75 DIC objective has no DIC prism
  • Condenser DIC prism is DIC N1 while objective requires N2
  • Check Stage top tilt adjustment procedure
  • Check Liquid light guide transmittance
  • 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 waterbath 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%
  • 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 wikipedia
  • 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

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

  • 20x/0.5 Air Ph1
  • 60x/1.4 Oil DIC WD 0.13
  • 100x/1.45 Oil Ph3 WD 0.13
  • 100x/1.45 Oil DIC WD 0.13
  • Empty
  • 20x/0.75 Air DIC

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 3cm 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 CMOS Monochrome Camera 2048 x 2048 pixels, 16-bit, 30fps at full frame Serial 101081

Workstation

  • HP Z440 Workstation
  • Intel Xeon E5-1620 v4 @ 3.5GHz
  • RAM 32 GB DDR4 2400 MHz ECC (4 x 8 GB)
  • OS 500GB SSD 550 MB/s
  • 4TB HD Data Storage (2 x 2 TB spanned volume) 170 MB/s
  • Video Card nVidia GTX 1080 8GB DDR5 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

Consumables

  • CO2 Tank
  • N2 Tank

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 acqusition

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 unlabeled 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 warmup the incubation chamber faster.

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
  • Immerge 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 20 minutes for the blank to reach 37°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 reach 33°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

Core Facilities CIB               Structural Biology    |    Cytometry    |    Microscopy    |    Histology

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