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Nikon Ti2-Fura microscope

Desmarais Building, Room 2236
Instrument awarded to the CI2B by the Canadian Foundation for Innovation (CFI)
Advanced Microscope Tier 1 usage price

 

Applications

  • Widefield
    • Brightfield

    • Fluorescence

  • Live imaging
  • Calcium ratiometric imaging



Light sources

  • LED lamp for transmitted light

  • CoolLed pE340-fura

  • Lumencor Spectra

Objectives

  1. 4x/0.2 Air
  2. 10x/0.45 Air
  3. 20x/0.75 Air
  4. 40x
  5. 60x/1.4 Oil DIC WD 0.13
  6. Empty
PositionNameBrandFull nameIdentifierWorking distance (mm)Transmittance
(% [nm])		TechniquesCover glass thickness (mm)
14x/0.2
Air		Nikon

4x/0.2 Air
CFI Plan Apochromat Lambda

MRD00045

20.0>80% [400-1000]BF, Fluo0.17
210x/0.45
Air		Nikon

10x/0.45 Air
CFI Plan Apochromat Lambda

MRD00105

4.0

 DIC N1
320x/0.75
Air		Nikon20x/0.75 Air
CFI Plan Apochromat Lambda		MRD00205

1.0

>80% [400-950]BF, Pol, DIC, Fluo
DIC N2		0.17
440x/0.75
Air		Nikon40x/0.75 Air
Plan Fluor 		MRH00400

0.72

 

5

60x/1.4
Oil

Nikon60x/1.4 Oil
CFI Plan Apochromat Lambda		MRD016050.13>80% [475-725]BF, Pol, DIC, Fluo
DIC N2
6

Empty





 

Filters

  1. DAPI

  2. GFP

  3. Cy3

  4. Cy5
  5. Fura
PositionNameBrandIDExcitation 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
3Cy3






4Cy5Semrock

Cy5-5070A

617/55x
[590-645]		652LP697/77m
[659-736]		Nikon ID 96376
5Fura






6Empty






Detector

  • Photometrics Prime 95B 25mm 

Camera

Photometrics Prime 95B

Sensor Type

Back-illuminated sCMOS

Sensor Category

Monochrome

Nb Pixels

2.6 M

Pixel Layout

1608 x 1608

Pixel size

11.0 um

Sensor size

17.7 mm x 17.7 mm 

Sensor diagonal

25 mm

Bit depth

16-bit
Speed at full resolution30 images/s

Max QE

95 %
Readout noise1.6 e⁻

Cooling

-20°C Air

Dark Current

0.55 e⁻/pixel/sec

Full well capacity

80 000 e-

Dynamic Range

1: 50 000

Interface

USB 3.0

Mount

F-mount

  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 left of the computer

  4. When using the instrument for the first time, it is necessary to import the microscope configuration before starting the software. See the First Use section below.

  5. Start-up 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 (ask for support if you are not sure).

  1. If open, close NIS-Elements and wait until it is completely closed (up to 30 seconds)
  2. On your 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-Fura_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. Save your data
  2. Close NIS-Elements software
  3. Transfer your data to the D: drive (Data Storage) or to your external drive and delete it from the local C: drive
  4. Turn off the computer
  5. If the oil objective was used, clean it with lens cleaner and lens paper (not Kimwipes)
  6. Wait until the Spectra has cooled down and turn off the microscope power bar (#2)
  7. Cover the microscope

Important Reminders

  • Collect your samples, especially those in the microscope
  • Leave the microscope and workspace clean
  • Files can be saved temporarily (during acquisition) to local C: drive (desktop)
  • At the end of each session, copy your data to your external drive and delete it from local C: drive
  • You can store your files on the D: drive (Data Storage). If you do, please create one folder per laboratory using the principal investigator's last name. Inside, create a folder per user using the following nomenclature (First Name_Last Name).

Important

In any case, do not store your files on the C: drive.

When using the microscope for the first time, you need to import the microscope settings into the software. You will usually do this during the training session.
This procedure can also be performed if something is not working properly and if you want to reset the software to its original settings.

This process will delete all experiment protocols and reset the software to the original settings for this specific microscope.

  1. If open, close NIS-Elements and wait until it is completely closed (up to 30 seconds)
  2. On your Desktop open the Softwares folder
  3. Open NIS Settings Utility
  4. Click on the Import tab
  5. Click on Browse
  6. Navigate to your Desktop
  7. Select the file Nikon Ti2-Fura settings for NIS.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
  • Fix camera adapter
  • Purchase 3mm adapter for Lapp
  • Purchase multiband pass cube for Spectra
  • Fix stage inserts
  • Add Stage insert screws
  • Complete installation
  • Complete cleaning

Stand

  • Nikon Ti2-E inverted  Serial System

Light sources

  • Transmitted LED light
    • ND filter:  TBD
  • CoolLED pE340-Fura Serial
  • Lumencor Spectra Serial Serial

Condenser

  • Manual condenser Serial

  • Lens LWD NA 0.52

  • Filter turret 7 manual positions

    1. ND TBD

    2. Empty
    3. Empty
    4. Empty
    5. Empty

Objectives

  • 4x/0.2 Air WD 20
  • 10x
  • 20x/0.75 Air DIC WD 1.0
  • 40x
  • 60x/1.4 Oil DIC WD 0.13

Stage

  • Motorized stage Encoded  Serial
  • Remote control joystick Ti2-S-JS Serial 
  • Inserts
    • Combo slide 3cm dish Ti2-S-HU with tilt adjustment (no incubation)

Filters

  1. DAPI Cube TBD
  2. GFP Cube TBD
  3. Cy3 Cube TBD
  4. Cy5 Cube TBD

Detector

  • Photometric Prime 25mm CMOS Monochrome Camera x 2048 pixels, 16-bit, 30fps at full resolution Serial 

Workstation

  • HP Z440 Workstation
  • Intel Xeon E5-1620 v4 @ 3.5GHz
  • RAM 32 GB DDR4 2400 MHz ECC (4 x 8 GB)
  • OS 500 GB SSD 530 MB/s
  • 4 TB HD Data Storage (2 x 2 TB spanned volume) 130 MB/s
  • Video Card nVidia GTX 1080 8GB DDR5 dedicated memory
  • Monitor HP Z24i display 24' 1920x1200
  • Software NIS-Elements AR v5.02

Consumables

  • Liquid Light Guide


Troubleshooting

This can happen when the acquisition software is turned on before the camera has fully initialized.

  • Turn off NIS-Elements
  • Ensure the Initialize light at the back of the camera no longer blinks
  • Start NIS-Elements

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

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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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