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 desk between the microscope and the computer
  4. 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
  5. 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-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. On the microscope, gently push the transmitted light arm backward

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

  3. If not done already, 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 parafocal, focusing with le safest objective 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 Z 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: Use the Display button to navigate the display menu
  • In NIS Elements: Ti2 Pad Tab under Z value

It is possible to scan an overview image to then easily navigate your sample. It is strongly recommended to use the lowest magnification objective and whenever possible use the BF optical configuration not to bleach your sample.

After completing the initial focus:

  1. In NIS Elements, click on the desired optical configuration (BF, DAPI, GFP, etc.)

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

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

  4. Using the Joystick navigate to the top left corner of your sample
  5. In the the XYZ overview click + to add a point and save this position
  6. Using the Joystick navigate to the bottom right corner of your sample
  7. In the the XYZ overview click + to add a point and save this position
  8. In the XYZ Overview right click and under Large Image Area select Define Area
  9. Draw a rectangle around the two points
  10. Right click again on the created region of interest and select scan preview
  11. Wait until the preview acquisition is completed

You can now directly click on the overview to navigate your sample !


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 air objective (10x, 20x, 40x)

    The 20x 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 Z 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 Z 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 Z to move the objectives to a safe position

  2. Click on the desired immersion objective (60x)

    The 60x is the best immersion lens because it has the highest numerical aperture (1.4).

  1. Remove the test slide

  2. Place a single drop of oil on the objective

  3. Place your sample

  4. Click Escape Z again to return the objectives to their normal position

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

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

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

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

  9. 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 click Escape Z to place the objectives in a safe position

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

  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


  • Fix camera adapter
  • Purchase 3mm adapter for Lapp
  • Purchase multiband pass cube for Spectra
  • Fix stage inserts
  • 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
  5. Fura

Detector

  • Photometric Prime95B 25mm Serial A18C203010

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

Manuals


Anti-vibration Table

  • TMC #63-42352-03 Serial 218043

Troubleshooting

This can happen if the acquisition software is turned on before the camera has fully initialized. The software should be started after 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