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Nikon Ti2-Fura microscope

Desmarais Building, Room 2236
Advanced Microscope Tier 1 usage price
Instrument awarded to the CI2B by the Canadian Foundation for Innovation (CFI #37439) in 2017

 

Applications

  • Inverted microscope
  • Widefield imaging
    • Brightfield

    • Fluorescence

  • Live imaging
  • Calcium ratiometric imaging
  • High-throughput imaging

Description

Light sources

  • LED lamp for transmitted light

  • CoolLed pE340-fura

    ChannelSourceFilterFluorophoreNominal Power (mW)Measured Power (mW)
    1340nmBP340/20

    Fura

    ?

    		TBD
    2380nmBP380/20

    Fura

    		?

    TBD

    3435-645nm-
    		?

    TBD

  • Lumencor Spectra

    ChannelSourceFilterBandwithFluorophoreNominal Power (mW)Measured Power (mW)
    Violet

    390

    390/22

    [380-400]

    DAPI, Hoechst, coumarins

    		236
    Blue

    437

    		437/26[424-450]CFP, Alexa Fluor 430, Pacific Blue, BFP301
    Cyan

    473

    		473/28[459-487]GFP, FITC, Alexa Fluor 488, Oregon Green, SYBR Green179
    Teal

    512

    		512/18[503-521]GFP (secondaire), YFP (partiellement), Alexa Fluor 514, SYTO dyes60.6
    Green/Yellow542

    542/30

    [527-557]

    Alexa Fluor 546, Cy3, TRITC

    		355

    Green/Yellow

    		575575/30

    [560-590]

    mCherry, DsRed, Alexa Fluor 568, Texas Red

    		293
    Red

    631

    		631/28

    [617-645]

    Alexa Fluor 633, Cy5, ATTO 647, mPlum, Alexa Fluor 647

    		243

Objectives

  1. 4x/0.2 Air
  2. 10x/0.45 Air
  3. 20x/0.75 Air
  4. 40x/0.75 Air
  5. 60x/1.4 Oil
  6. -
    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
    		BF, Fluo0.17
    210x/0.45
    Air    		Nikon

    10x/0.45 Air
    CFI Plan Apochromat Lambda

    		MRD00105

    4.0

    		 BF, DIC N1, Fluo0.17
    320x/0.75
    Air    		Nikon20x/0.75 Air
    CFI Plan Apochromat Lambda    		MRD00205

    1.0


    		BF, DIC N2, Fluo0.17
    440x/0.75
    Air    		Nikon40x/0.75 Air
    Plan Fluor     		MRH00400

    0.72

    		 
    		0.17
    5

    60x/1.4
    Oil

    		Nikon60x/1.4 Oil
    CFI Plan Apochromat Lambda    		MRD016050.13
    		BF, DIC N2, Fluo0.17
    6

    -

    		----- --

Filters

  1. DAPI

  2. GFP

  3. Cy3

  4. Cy5
  5. Fura
  6. -
PositionNameBrandIDExcitation FilterDichroic mirrorEmission FilterComments
1DAPISemrock96370 M352024-409LP447/60Semrock Brightline 96370 M352024 DAPI-3060A
No excitation filter
2GFPSemrockGFP-4050B

466/40x

495LP

525/50mSemrock Brightline 96372 M380163-17 GFP-4050B
3Cy3Semrock

LED-TRITC-A

554/23573LP609/54Semrock Brightline 96374 M380162-10 LED-TRITC-A
4Cy5Semrock

Cy5-5070A

618/50652LP698/70

Semrock Brightline 96376 M351081 23 Cy5-5070A

5FuraChroma

79001-ET-Fura-2

-400LP510/80

Chroma Fura 77074017 79001-ET-Fura2 C189116
Smaller cube (not 32mm). No Excitation Filter.

6--

-

----


Detector

  • Photometrics Prime 95B 25mm 

Camera

Photometrics Prime 95B 25mm

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

User Guide

  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 create a preview 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 with the coverslip facing toward the objective

  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

Log

  • Fix camera adapter
  • Purchase multiband pass cube for Spectra
  • Fix stage inserts Ti2 S HU
  • Purchase Multiwell plate insert TI2 S HW
  • Replace LLG Spectra
  • Add HCA Jobs and Plates user guide
  • Installation Spectra
  • Installation LAPP
  • Complete installation
  • Complete cleaning

Technical Datasheet

Stand

  • Nikon Ti2-E inverted Serial 540749
  • Pillar for transmitted illumination Ti2-D-PD Serial 599941
  • Manual condenser Turret TC-C-TC
  • Perfect Focus Unit with motorized nosepiece Ti2-N-ND-P Serial 128285
  • 6 positions motorized epi filter turret Ti2-F-FLT-E Serial 510911
  • EPI-FL module TI2-LA-FLL Serial 547589
  • Ti2-LAPP System Ti2-LA-BM-E Serial 548536
  • Camera adapter Ti2-BDTV2
  • Ti2 Controller Ti2-CTRE Serial 451316

Light sources

  • LED Lamphouse for dia illumination Ti2-D-LHLED Serial 557988
  • CoolLED pE340-Fura Serial AY1035
  • Lumencor Spectra7-API-IB Serial 3719 with LLG adapter 82-10012 Rev B Applied precision part number 34-100926-000

Condenser

  • LWD Condenser lens (O.D.=30 mm, NA=0.52)

  • Filter turret 7 manual positions

    1. Neutral Density ND

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

Objectives

  • 4x/0.2 MRD00045
  • 10x/0.45 MRD00105
  • 20x/0.75 MRD00205
  • 40x/0.75MRH00400
  • 60x/1.4 MRD01605
  • Empty

Stage

  • Motorized stage Encoded Ti2-S-SE-E Serial 960166
  • Remote control joystick Ti2-S-JS Serial 128408
  • Inserts
    • Combo slide 3cm dish Ti2-S-HU with tilt adjustment (no incubation)

Filters

  1. DAPI Cube Semrock Brightline 96370 M352024 1 No excitation filter
  2. GFP Cube Semrock Brightline 96372 M380163-17 GFP-4050B Ex 466/40 Em 525/50 Di 495
  3. Cy3 Cube Semrock Brightline 96374 M380162-10 LED-TRITC-A Ex 554/23 Em 609/54 Di 573
  4. Cy5 Cube Semrock Brightline 96376 M351081 23
  5. Fura77074017 79001 ET Fura2 C189116 (smaller cube not 25mm) No Excitation Filter
  6. Empty

Detector

  • Photometric Prime95B 25mm Serial A18C203010

Workstation

  • HP Z440 Workstation
  • I155439-CIB
  • Intel Xeon E5-1620 v4 @ 3.5GHz
  • Motherboard HP 212B Chipset Intel C612
  • BIOS M60 V02.61 2023-03-23
  • RAM 32 GB DDR4 1200 MHz ECC (4 x 8 GB)
  • OS 1 TB NVMe 5500 MB/s via PCie x4 to M2 Adapter Startech PEX4M2E
  • 4 TB HD Data Storage (2 x 2 TB spanned volume) 212 MB/s
  • Video Card nVidia Quadro P4000 8GB GDDR5 5.3 TFLOPS FP32
  • Monitor HP Z27n display 27' 2560x1440
  • Software NIS-Elements AR v5.2 HASP 53A125C5  NIS Elements AR
    • ND (6 dimensions)
    • Quick Piezo Z
    • Quick MultiExcitation
    • Shutter
    • Wavelength Switcher
    • Hight content
    • JOBS
    • General Analysis 
    • JOBS View
    • Remote DB for JOBS
    • General Analysis 3

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

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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"