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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 

Filter nameFilter IDFilter typeExcitation wavelengths (nm)Compatible fluorophores
V

395/25

Bandpass[382-407]

DAPI, Hoechst

B

440/20

Bandpass[430-450]CFP
C

470/24

Bandpass[458-482]FITC, GFP
NT

510/25

Bandpass[497-522]YFP
GY550/15Bandpass

[542-557]

TRITC, Cy3

GY (Storage)

575/25Bandpass[562-587]

mCherry

R

640/30

Bandpass[625-655]

Cy5

  • 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 GY position)

    5. DIC Analyzer
    6. DAPI/CFP/YFP/mCherry (requires mCherry filter in lumencor GY 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

Nikon

C182279

560/55x [532-588]

595LP645/75m [607-682]77074160 Custom Quad C182279
5

DIC Analyzer

NikonTi2-C-DICACLNone
395/25;470/24;550/15;640/3
6

CFP\YFP\mCherry

NikonC1997767None459/526/596

475/543/702

C1997767

  • 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 water bath is 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.

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.

Light path schematics.pdf

  • Add label on gas bottle
  • Add line mark on humidifer
  • Add light path to wiki
  • Place label numbers following user user guide
  • Check Condenser filter if label match
  • 20x DIC objective has no DIC prism
  • Condenser DIC prism is DIC N1 while objective requires N2
  • Print a memo about objective DIC vs Ph and Oil vs air
  • 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
    • 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
PositionNameBrandIDMagnificationNumerical ApertureImmersionTypeWorking distance (mm)Transmittance
(% [nm])		TechniqueCover glass thickness (mm)
120x/0.5 Air Ph1Nikon

20x/0.5 Air Plan Fluor Ph1

20x0.5AirPlan Fluor2.1>80% [400-750]BF, Pol, PhC, Fluo0.17
2

60x/1.4 Oil DIC

Nikon60x/1.4 Oil Plan Apo Lambda DIC N260x

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 Ph3

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 N2

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 N220x0.75AirPlan Apo Lambda

1.0

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

Stage

  • Motorized stage Ti2 SHU compatible Serial 127808
  • Remote control joystick Ti2-S-JS Serial 127976
  • Inserts
    • Combo slide and 3cm dish with tilt adjustment insert
    • Multi-well plate insert

Filters

  1. Ex 383-408 DAPI-U DM 425 BA 435-485
  2. Semrock 96372 M349727 17
  3. 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. 7707\4656 CFP\YFP\mCherry XT C197767

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

Liquid forming in the incubation chamber

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
  • 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

Can I use this microscope to look at cell in a dish?

  • 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


Core Facilities CIB               Structural Biology    |    Cytometry    |    Microscopy    |    Histology


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