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_Micro-head-en
_Micro-head-en

Button Hyperlink
iconbuild
titleFrançais
typeprimary
urlhttps://wiki.umontreal.ca/display/Microscopie/Nikon+Ti2-E

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



Available manuals
Tabs Container
directionhorizontal
Tabs Page
idDescription
titleDescription

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
Expand
titleComplete specifications
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
Expand
titleComplete specifications
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 

Expand
titleComplete specifications

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

View file
namePhotometrics_Prime-95B_Datasheet.pdf
height250
View file
namePhotometrics_Prime-95B_Manual.pdf
height250

Tabs Page
idUser Guide
titleUser Guide
UI Expand
expandedtrue
titleStart-up
  1. If not already done,
    2.  
  2. turn on the computer (#1) and log in to Windows using your UdeM credentials
  3. Remove the dust cover from the microscope
  4. Turn on the microscope power bar (#2) on
    5. the left of
  5. the desk between the microscope and the computer
  6. When using the instrument for the first time, it is necessary to import the microscope configuration
    7. before starting
  7. into the software. See
    8. the 
  8. the First Use section below
    9. .
  9. before starting the software
  10. Start
    11. -up
  11. NIS-Elements
UI Expand
expandedtrue
titleFirst Use

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. 

Note

Running this procedure will erase all your experiment protocols and reset the software to its original settings (ask for support if . If you are not sure), ask for support.

  1. If NIS-Elements is open, close

    2.  NIS-Elements 

  2. it and wait until it

    3. is

  3. has completely

    4. closed

  4. shut down (this may take up to 30 seconds)

  5. On

    6. your Desktop open the Softwares folder

  6. the Desktop, open the Softwares folder

    7. Open 
  7. Open
  8. NIS Settings Utility
  9. Click on the
    10.  
  10. Import
    11.  
  11. tab
  12. Click on
    13.  
  13. Browse
  14. Navigate to your C:\Users\Public\Documents
  15. Select the file
    16.  
  16. Nikon_Ti2-Fura_NIS Settings.bin
  17. Click
    18.  
  18. Select
  19. Select all items
  20. Click
    21.  
  21. Import
  22. Click
    23.  
  23. OK
  24. Close the
    25.  
  25. NIS Settings Utility
  26. You can now reopen NIS-Elements
UI Expand
titleLoading samples

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.

UI Expand
title
Shutdown
Initial Focus

  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

    Info
    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

    Tip

    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
Tip

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
UI Expand
titleScanning an overview

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 !


UI Expand
titleSecondary focus
Warning

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

UI Expand
titleFocusing with air objectives

After completing the initial focus:

  1. In NIS-Elements, click on the desired air objective (10x, 20x, 40x)

    Info

    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!

UI Expand
titleFocusing with oil objectives

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)

    Info

    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!

  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
Note
titleImportant Reminders
  • Collect your samples, especially those in the microscope
    • Leave the microscope and workspace clean


    UI Expand
    titleStorage management
    • Files can be saved temporarily (during acquisition)
      • to
    • on the local C: drive (desktop)
      • elete
    • At the end of each session, copy your data to your external drive and
      • d
    • delete it from the local C: drive
    • You can store your files on the D: drive (Data Storage). If you do, please create
      • one
    • a folder per laboratory using the principal investigator
      • 's
    • last name.
      • Inside
    • Within, create
      • a
    • one folder per user
      • using the following nomenclature (First Name_Last Name
    • (Firstname_Lastname).
    title
    Note
    Important

    In any case, do not store your files on should be removed from 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.
    UI Expand
    titleFirst use
    Anchor
    FirstUseFirstUse
    Note

    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
    Tabs Page
    idManuals
    titleManuals
    Shutdown
    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
    Note
    • Take back your samples including ones in the microscope
    • Leave the microscope and the working area clean

    Tabs Page
    id
    Tabs Page
    idLog
    titleLog


    UI Expand
    expandedtrue
    titleTo do
    • Fix camera adapter
    • Purchase 3mm adapter for Lapp
    • Purchase multiband pass cube for Spectra
    • Fix stage insertsAdd Stage insert screws
    UI Expand
    title2025-03-10 Insallation Desmarais 2236
    • Complete installation
    • Complete cleaning
    Tabs Page
    idTechnical Datasheet
    titleTechnical Datasheet

    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 Prime 25mm CMOS Monochrome Camera x 2048 pixels, 16-bit, 30fps at full resolution Serial 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
    Tabs Page
    idFAQ
    titleTroubleshooting & FAQ

    Troubleshooting

    UI Expand
    titleCamera Driver Failed

    This can happen when 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

    UI Expand
    titleCan 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
    • For long timelapse, be aware of photo-toxicity


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