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Include Page
_Micro-head-en
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Button Hyperlink
iconbuild
titleFrançais
typeprimary
urlhttps://wiki.umontreal.ca/display/Microscopie/Nikon + Ti2-E

Tabs Container
idInstrument Tabs
titleNikon Ti2-E
directionhorizontal
Tabs Page
idDescription
titleDescription

Fura

Nikon Ti2-Fura

inverted

microscope

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

Advanced Microscope Tier 1 usage price
in 2017

 

Applications

Transmitted light, Bright-field
  • Inverted microscope
  • Widefield imaging
    • Brightfield

    • Fluorescence

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

Image Added

Image Added

Tabs Container
directionhorizontal
Tabs Page
idDescription
titleDescription

Description

Light sources

  • LED lamp for transmitted light

  • CoolLed pE340-fura

    Expand
    titleComplete specifications
    ChannelSourceFilterFluorophoreNominal Power (mW)Measured Power (mW)
    1340nmBP340/20

    Fura

    ?

    		TBD
    2380nmBP380/20

    Fura

    		?

    TBD

    3435-645nm-
    		?

    TBD

    Image Added

    View file
    nameCoolLED_pE-340fura_User-Manual.pdf
    height250
    View file
    nameCoolLED_pE-340fura_Quick Start Guidepdf.pdf
    height250
    View file
    nameCoolLED_pE-340fura_Troubleshooting Guide.pdf
    height250
    View file
    nameCoolLED_pE-340fura_Diagram.pdf
    height250
    View file
    nameCoolLED_pE-340fura_Datasheet.pdf
    height250

  • Lumencor Spectra

    Expand
    titleComplete specifications
    ChannelSourceFilterBandwithFluorophoreNominal Power (mW)Measured Power (mW)
    Violet

    390

    390/22

    [379-401]

    DAPI, Hoechst, coumarins

    		236
    Blue

    437

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

    475

    		485/20[475-495]GFP, FITC, Alexa Fluor 488, Oregon Green, SYBR Green179
    Teal

    512

    		512/18[503-521]YFP, Alexa Fluor 514, SYTO dyes, Rhodamine 12360.6
    Green/Yellow542

    542/30

    [527-557]

    Rhodamine, Alexa Fluor 546, 555, 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

    View file
    nameLumencor_Spectra_User-Manual.pdf
    height250
    View file
    nameLumencor_Specra_Certificate of Conformance.pdf
    height250
    Image Added
    View file
    nameLumencor_Spectra X_Manual.pdf
    height250

Objectives

  1. 4x/0.2 Air
WD 20
  2. 10x/0.45 Air
  3. 20x/0.75 Air
DIC WD 1.0
  2. 40x/0.75 Air
  3. 60x/1.4 Oil
DIC WD 0.13
Empty
  1. -
    Expand
    title
Objectives complete
  1. Complete specifications
    PositionNameBrandFull nameIdentifierWorking distance (mm)Transmittance
    (% [nm])    		TechniquesCover glass thickness (mm)
    14x/0.2
    Air    		Nikon

    4x/0.2 Air
    CFI Plan

Apo

  1. Apochromat Lambda

    MRD00045

    		20
>80% [400-1000]
  1. .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
DIC
  1. Nikon20x/0.75 Air
    CFI Plan
Apo
  1. Apochromat Lambda
DIC N2
  1. MRD00205

    1.0

>80% [400-950]

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

    0.72

    		 
    		0.17
    5

    60x/1.4
    Oil

DIC

  		1. Nikon60x/1.4 Oil
    CFI Plan
Apo
  1. Apochromat Lambda
DIC N2
  1. MRD016050.13
>80% [475-725]

  		1. BF,
Pol,
  1. DIC N2, Fluo0.17
    6

    -

    		----- --

Filters

  1. DAPI

  2. GFP

  3. Cy3

  4. Cy5
  5. Fura
  6. QuadBand DAPI/FITC/TRITC/Cy5
Expand
title
Filters complete
Complete specifications
PositionNameBrandIDExcitation FilterDichroic mirrorEmission FilterComments
1DAPI
NikonDAPI-U HQ395/25x
[383-408]425LP460/50m
[435-485]
Semrock96370 M352024-409LP447/60Semrock Brightline 96370 M352024 DAPI-3060A
No excitation filter
C-FL-C DAPI-U HQ
2GFPSemrockGFP-4050B
-000

466/40x

[446-486]3Cy3

495LP

525/50m
[500-550]Nikon ID 96372
Semrock 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.

6QuadBandSemrock

-

Whitin Spectra

390/22 [379-401]
485/20 [475-495] 
540/30 [527-557]
631/28 [617

/55x
[590-645]652LP697/77m
[659-736]Nikon ID 963765Fura

  • Detector

    • Photometrics Prime 95B 25mm CMOS Monochrome Camera  x  pixels, 16-bit, 30 images/s at full frame (100 images/s at full frame with camera link connector)

Tabs Page

-645]

FF409/493/573/652-Di02
Reflection - Transmission
350-404nm - 414-450nm
461-487nm - 499-530nm
543-566nm - 580-611nm
626-644nm - 659-850nm		F01-432/515/595/730-32 
432/36 [414-450]
515/30 [499-530] 
595/31 [580-611]
730/139 [661-800]

DAPI excitation can be improved by 375/40
FITC excitation should be replaced 474/24
TRITC excitation should be replaced with 554/22
Cy5 excitation filter can be improved with a 635/18


Detector

  • Photometrics Prime 95B 25mm 

Expand
titleComplete specifications

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

Image Added

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

Tabs Page
titleUser Guide
idUser Guidetitle

User Guide

UI Expand
expandedtrue
title
StartupTurn
Start-up
  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
Use your UdeM credentials to log in to Windows
  1. 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
  2. Start NIS-Elements
UI Expand
titleFirst Use

When using the instrument for the first time, it is necessary

InfoThe first time you use the instrument, you need Start the software

to import the microscope

settings into the software. To do this follow the instructions First use protocol.

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. 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 
  1. NIS-Elements
UI Expand
title
Shutdown
  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
Loading 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
titleInitial 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
titleCreate a preview

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 !

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 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!

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 elete
    • on the local C: drive (desktop)
    • 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).
    Note
    titleImportant

    In any case,

    do not store

    your files

    on

    should be removed from the C: drive.

    UI Expand
    title
    First use AnchorFirstUseFirstUseWhen 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.
    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
    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
    titleLog
    Tabs Page
    idManuals
    titleManuals
    Available manuals Tabs PageidLogtitle

    Log

    UI Expand
    expandedtrue
    titleTo do
    • Fix camera
    adapterPurchase 3mm
    • adapter
    for Lapp
    • Purchase multiband pass cube for Spectra
    • Fix stage inserts Ti2 S HU
    Add Stage insert screws
    • Replace LLG Spectra
    • Add HCA Jobs and Plates user guide
    UI Expand
    title2026-02-24 Installation multiwell insert
    • Purchase Multiwell plate insert TI2-S-HW

    Image Added

    UI Expand
    title2025-10-09 Installation Spectra
    • Installation Spectra
    • Installation LAPP
    UI Expand
    title2025-03-10 Insallation Desmarais 2236
    • Complete installation
    • Complete cleaning
    Tabs Page
    id
    titleTechnical Datasheet
    title

    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-
    E inverted  Serial System
    • 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

  • Transmitted LED light
    • ND filter:  TBD
    • LED Lamphouse for dia illumination Ti2-D-LHLED Serial 557988
    • CoolLED pE340-Fura Serial AY1035
    • Lumencor
    Spectra Serial SerialLens LWD NA
    • Spectra7-API-IB Serial 3719 with LLG adapter 82-10012 Rev B Applied precision part number 34-100926-000

    Condenser

  • Manual condenser Serial

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

    • Filter turret 7 manual positions

      1. Neutral Density ND

    TBD

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

    Objectives

    • 4x/0.2
    Air WD 20
    • MRD00045
    • 10x/0.45 MRD00105
    • 20x/0.75
    Air DIC WD 1.0
    • MRD00205
    • 40x/0.75MRH00400
    • 60x/1.4
    Oil DIC WD 0.13
    • MRD01605
    • Empty

    Stage

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

    Filters

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

    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
    • 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 2400
    • GB DDR4 1200 MHz ECC (4 x 8 GB)
    • OS
    500 GB SSD 530
    • 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)
    130
    • 212 MB/s
    • Video Card nVidia
    GTX 1080 8GB DDR5 dedicated memory
    • Quadro P4000 8GB GDDR5 5.3 TFLOPS FP32
    • Monitor HP Z27n display 27' 2560x1440
    Monitor HP Z24i display 24' 1920x1200
    • Software NIS-Elements AR v5.
    02
    • 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

    View file
    nameLumencor_Spectra X_Manual.pdf
    height250
    View file
    nameLumencor_Specra_Certificate of Conformance.pdf
    height250
    View file
    nameCoolLED_pE-340fura_Datasheet.pdf
    height250
    View file
    nameCoolLED_pE-340fura_Diagram.pdf
    height250
    View file
    nameCoolLED_pE-340fura_Troubleshooting Guide

    Tabs PageidFAQ

    .pdf
    height250
    View file
    nameCoolLED_pE-340fura_Quick Start Guidepdf.pdf
    height250
    Image AddedImage Added
    View file
    nameCoolLED_pE-340fura_Brochure.pdf
    height250
    View file
    nameCoolLED_pE-340fura_User-Manual.pdf
    height250
    View file
    namePhotometrics_Prime-95B_Manual.pdf
    height250
    View file
    namePhotometrics_Prime-95B_Datasheet.pdf
    height250
    View file
    nameNational Instruments_NIDAQ_Triggering Guide for NIS-Elements.pdf
    height250

    Anti-vibration Table

    • TMC #63-42352-03 Serial 218043
    Tabs Page
    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
    Tabs Container
    idDemo Image
    titleNikon Ti2-E
    directionhorizontal
    Tabs Page
    idDemo Image
    titleDemo Image

    Image Removed

    Image Removed

    Include Page
    Plateformes:_Plat-foot-en
    Plateformes:_Plat-foot-en