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Nikon TE2000-E inverted microscope

Bombardier Building, Room 3132

Usage Price Advanced Microscope Tier 1

Instrument awarded to Dr. Steve Michnick by the Canadian Foundation for Innovation (CFI)

  • Transmitted light
  • Phase Contrast
  • Interference Contrast (DIC)
  • Fluorescence
  • Widefield imaging
  • Live imaging
  • Time-lapse imaging

Light sources

  • Halogen lamp for transmitted light

  • X-Cite exacte for fluorescence

Emission (nm)

Nominal power (mW)

Objectives

  1. 4x/0.1 Air
  2. 10x/0.30 Ph1 Air
  3. 20x/0.45 Ph1 Air
  4. 40x/0.95 Air
  5. 60x/1.4 Ph3 Oil
  6. 100x/1.45 Oil
PositionNameBrandFull nameIdentifierMagnificationNumerical apertureImmersionWorking distance (mm)Transmittance
(% [nm])		ApplicationsCover glass thickness (mm)
14x/0.10 AirNikon4x/0.1 Plan

MRL00042

4x0.1Air30>80% [400-900]BF, Fluo-
210x/0.30 Ph1 AurNikon10x/0.30 Plan Fluor Ph1 DL

MRH20101
MRH20105

10x0.3Air15.2>80% [440-700]BF, PhC, Fluo1.2 !
320x/0.45 Ph1 AirNikon20x/0.45 Plan Fluor ELWD Ph1 ADL

MRH48230
MRH48220

20x0.45Air7.4>80% [350-870]BF, PhC, Fluo0-2
440x/0.95 AirNikon40x/0.95 Plan Apo DIC M/N2

MRD00400
MRD70470

40x0.95Air0.14>80% [440-760]BF, Fluo0.11-0.23
560x/1.4 Ph3 OilNikon60x/1.4 Plan Apo
Ph3 DM

MRD31600

60x1.4Oil0.21 BF, PhC, Fluo0.17
6100x/1.45 OilNikon100x/1.45 Plan Apo Lambda  OFN25 DIC N2

MRD01905
MRD71970

100x1.45Oil0.13>80% [460-720]BF, Fluo0.17
-40x/0.60 Ph2 AirNikonPlan Fluor 40x/0.60 Ph2 DIC M ELWD 0-2

MRH48430

40x0.6Air2.7-3.7>80% [350-850]BF, PhC, Fluo0-2
-40x/0.60 AirNikonS Plan Fluor 40x/0.6 ELWD DIC N1

MRH08430

40x0.6Air2.8-3.6>80% [350-850]BF, Fluo0-2
-40x/0.90 AirNikonS Fluor 40x/0.90 DIC M/N2

NA

40x

0.9

Air0.3NA
BF, Fluo0.11-0.23
-60x/0.70 AirNikonPlan Fluor 60x/0.70 ELWD DIC M/N1

MRH08630

40x

0.7


Air1.5-2.1>80% [380-880]BF, Fluo0.5-1.5
-60xA/1.40 OilNikonPlan Apo 60xA/1.40 Oil DIC H

MRD71670

60x1.4Oil0.21>80% [480-700]BF, Fluo0.17
-60x/1.40 OilNikonPlan Apo VC 60x/1.40 Oil DIC N2

MRD71670

60x1.4Oil0.13>80% [480-700]BF, Fluo0.17
-100x/0.85 AirNikonL Plan 100x/0.85 ODN 25

MUE35900

100x0.85Air0.85 
0-0.7
-100x/1.40 OilNikonPlan Apo 100x/1.4 Oil DIC H

MRD71970

100x1.4Oil0.13>80% [480-720]BF, Fluo0.17

 BF: Bright-field
PhC: Phase Contrast
DIC: Differential Interference Contrast
Fluo: Fluorescence

Filters

  1. Dichroic DAPI\FITC\TRITC

  2. Dichroic CFP\YFP\Cy5

  3. Dichroic CFP\YFP\mCherry

  4. DAPI
  5. Cy3n
  6. Cy5.5

Also available

  • CFP

  • FITC

  • YFP

  • Cy3.5

  • mCherry

  • Cy5

Filter cubes

PositionNameBrandIdentifierExcitationDichroicEmissionComments
1Dichroic
DAPI\FITC\TRITC		Chroma86013 v2
86013bs
To be used in combination with excitation and emission filters
2

Dichroic
CFP\YFP\Cy5

Chroma86008 v2


86008bs


To be used in combination with excitation and emission filters
3

Dichroic
CFP\YFP\mCherry

Chroma

89006 ET


69008bs
To be used in combination with excitation and emission filters
4DAPIChroma

31000 v2

AT350/50x

400dclp

D460/50m


5Cy3nChroma

SP102 v1

HQ546/11xQ557lp

HQ567/15m


6Cy5.5Chroma

41022

HQ665/45x

Q695lpHQ725/50m
-eCFPChroma

49001 ET

ET436/20x

T455lp

ET480/40m


-cGFPChroma

31044 v2

D436/20x

455dclp

D480/40m


-FITCChroma

41001

HQ480/40x

Q505lp

HQ535/50m


-YFPChroma

49003 ET

ET500/20x

T515lp

ET535/30m


-Cy3.5Chroma

SP103 v1

HQ581/10x

Q593lp

HQ617/40m


-mCherryChroma

49008 ET

ET560/40x

T585lpxr

ET630/75m


-Cy5Chroma

49006 ET

ET620/60x

T660lpxr

ET700/75m


Excitation filters

PositionNameBrandIdentifierExcitationDichroicEmission
1DAPIChroma86013 v2 Exc 1AT350/50x

86013bs

 		S457/17m
2

CFP

Chroma86008 v2 Exc 1

S430/25x

86008bs
69008bs

S465/30m
3FITCChroma86013 v2 Exc 2

S484/15x

86013bs

S517/30m

4

YFP

Chroma86008 v2 Exc 2

S510/20x

86008bs
69008bs

S550/50m

5TRITCChroma86013 v2 Exc 3S555/25x

86013bs

S605/40m

6

mCherry

Chroma89006 ET Exc 3

ET572/35x

69008bs

ET632/60m

7

Cy5

Chroma86008 v2 Exc 3

S622/36x

86008bs

S700/75m
8

Empty






Emission filters

PositionNameBrandIdentifierExcitationDichroicEmissionComments
2

CFP

Chroma86008 v2 Em 1

S430/25x

86008bs
69008bs

S465/30m
3FITCChroma86013 v2 Em 2

S484/15x

86013bs

S517/30m


4

YFP

SemrockFF01-550/49-26

S510/20x

86008bs
69008bs

F01-550/49-25


5TRITCChroma86013 v2 Em 3S555/25x

86013bs

S605/40m


6

mCherry

SemrockFF01-641/75-25

ET572/35x

69008bs

FF01-641/75-25


7

Cy5

Chroma86008 v2 Em 3

S622/36x

86008bs

S700/75m
8

Empty







9

Shutter







10

Shutter







Detector

  • Hamamatsu ORCA Flash v2 CMOS

    • Monochrome Camera

    • 2048 x 2048 pixels

    • Pixel  6.5 um x 6.5 um

    • 30 images/s at full frame (100 images/s at full frame with camera link connector)

    • Dynamic range 1 : 18000 16-bit 

    • Spectral response >40% between 400-850nm, Max 80% at 550nm

  1. If not already done, turn on the computer (#1) and use your UdeM credentials to log in to Windows
  2. Remove the cover from the microscope
  3. Turn on the X-Cite exacte light source (#2)
  4. Turn on the microscope power bar (#3)
  5. When using the software for the first time, it is necessary to import the microscope-specific settings before launching the acquisition software. Please refer to the First Use section below for instructions.
  6. Start NIS-Elements

When using the acquisition software for the first time, it is necessary to import the microscope-specific parameters into the software. This procedure is usually carried out during the training session. However, it is also possible to use it to reset the software if it is not displayed correctly, for example.

Please note, this procedure will delete all your experiment protocols and restore the software to its original settings.

  1. If open, close NIS-Elements and wait until it is completely closed
  2. On the Desktop open the folder Softwares
  3. Open NIS Settings Utility
  4. Click on the Import tab
  5. Click on Browse
  6. Navigate to your Documents
  7. Select the file NIS Settings for Nikon TE2000E.bin
  8. Click Select
  9. Select all items
  10. Click Import
  11. Click OK
  12. Close NIS Settings Utility
  13. You can then open NIS-Elements

This procedure puts the microscope in a safe configuration to load your sample. At the end the microscope will be ready for acquisition.

  1. If not already done, in NIS-Elements select 4x to select the 4x objective

    The 4x objective is the safest because it has the longest working distance (30mm). The sample will appear perfectly sharp long before the lens approaches it. It is recommended to always first focus with the safest lens. The objectives are parafocal, focusing with the safest objective will then allow you to easily find your sample with another objective. The 10x objective is also safe because its working distance is 15.2 mm.

  2. If not done already, press Escape to lower the objective to the lowest position
  3. Place the test slide on the microscope stage with the coverslip toward the objective

    Important

    Always use the test slide to perform the first focus.

  4. If necessary, move the stage so that the sample is centered on the objective
  5. In NIS-Elements, select the optical configuration BF vis or the desired fluorescence (DAPI vis, ...) to activate the configuration
  6. Adjust the focus with the main dial while looking through the eyepieces until the image is perfectly sharp

    The focus can be found at Z = mm). The Z value can be found in the NIS Software XYZ Position tab.


  7. In NIS-Elements, select the optical configuration Off to turn off the illumination

Important

First focus with the safest objective before selecting another lens and continuing with secondary focus.

  1. In NIS-Elements, click 10x, 20x or 40x to select the desired objective

    The 40x objective is the best Air objective because it has the greatest number of optical corrections (Plan Apochromat) and the largest numerical aperture (0.95).


  2. In NIS-Elements, select the optical configuration BF vis or the desired fluorescence (DAPI vis, ...) to activate the configuration
  3. Adjust the focus with the precision dial while looking through the eyepieces until the image is perfectly sharp 
  4. Select the optical configuration Off to turn off the illumination
  5. Your sample is ready for acquisition!

After performing the first focus, in NIS-Elements:

  1. In NIS, click Escape, to lower the objectives

  2. Click 60x Oil or 100x Oil to select the desired objective. 

    The 100x objective has the greatest resolution (NA 1.45 vs 1.4)

  3. Place a single drop of oil on the objective
  4. In NIS-Elements, click again Escape to return the objective to its original position
  5. Select the optical configuration BF vis or the desired fluorescence (DAPI vis, ...) to activate the configuration
  6. Adjust the focus with the precision dial while looking through the eyepieces until the image is perfectly sharp 
  7. Select the optical configuration Off to turn off the illumination
  8. Your sample is ready for acquisition!
  • 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.

  1. Save your data
  2. If the oil objective was used, clean it with lens cleaner and lens paper (not Kimwipes)
  3. Select the lowest magnification objective and press Escape to place the objectives in a safe position
  4. Close NIS-Elements
  5. Transfer your data to the D: drive (Data Storage) or to your external drive and delete it from the local C: drive
  6. Turn off the computer
  7. Turn off the X-Cite exacte light (#2)
  8. Turn off the microscope power bar (#3)
  9. Cover the microscope

Important Reminders

  • Collect your samples, especially those in the microscope
  • Leave the microscope and workspace clean
  • Perfect Focus avec LED de 780 nm et avec filtre dans le trajet lumineux LP 750 nm (inadéquat pour le Cy5.5)

  • En lumière transmise, pour que le PFS fonctionne, il faut insérer 2 filtres IR dans le trajet lumineux: celui du PFS et celui du statif près de la lampe halogène

  • Complete installation
  • Complete cleaning

Stand

  • Nikon TE2000-E inverted
  • Nikon T-HUBC Serial 750110
  • Nikon PFS T-PFS 810060
  • Nikon T-RCP Serial 750104

Light sources

  • Transmitted Halogen light Nikon TE2-PS100W Serial 505320 12V
    • GIF Filter
    • Diffusion
    • Infra-red
    • Neutral color
    • Transmitted light shutter uniblitz VCM D1 Serial A607295
  • X-Cite exacte Model XCT10A 200 W Seria;l XCT10A-0337
    • EXFO Collimator 810-00030
    • Prior Shutter HF201HT Serial 7332
    • Prior 8 position motorized excitation filter wheel 61817
    • Nikon Manual Neutral density filters ND8 ND4
    • Manual Fluorescence field diaphragm
    • Manual Fluorescence shutter

Condenser

  • Motorized condenser

  • Lens LWD NA 0.52

  • Manual Polorizer

  • Filter turret 5 motorized positions

    1. A BF

    2. DIC M
    3. Ph1
    4. Ph2
    5. Ph3

Objectives

  • See description

Stage

  • Prior Proscan 2 H30V4 Serial 62040
  • Remote control joystick Prior CS152v2 Serial R0772128
  •  Inserts
    • 1 slide
    • Multi-well plate
    • Adapter for 3cm dish

Filters

  • See description

Detector

  • Hamamtsu ORCA Flash V2 CMOS Model C11440-22CU Serial 002162 V2
  • Monochrome Camera 2048 x 2048 pixels
  • Pixel  6.5 um x 6.5 um
  • Force air cooled at -10C
  • Effective sensor area 13.312 mm (H) × 13.312 mm (V
  • Dark current 0.06 electron/pixel/s
  • Reading noise 1.6 electrons (r.m.s) at standard scan
  • Dynamic range 1 : 18 000
  • Spectral response >40% between 400-850nm. Max 80% at 550nm

Workstation

  • HP Z800 Workstation
  • 2 x Intel Xeon X5650 @ 2.67GHz
  • RAM 24 GB DDR3 666 MHz  (6 x 4 GB)
  • OS 500 GB SSD 530 MB/s
  • 3 TB HD Data Storage (2 TB + 1 TB spanned volume) 130 MB/s
  • Video Card AMD FirePro V5800 1 GB dedicated memory
  • Monitor Dell 2407EFP-HC 1900x1200 60Hz
  • Software NIS-Elements AR v4.20.02 Build 988 HASP ID 4683DFE6
  • Core NIS-AR 2.3x STANDARD SUA Activation date 2010-07-07 SUA paid 2x SUA Expiration date 2013-08-07
  • Modules SRM ,Filter Wheel, ND (6 dimensions), Shutter, Stage XY axis

Consumables

  • Liquid Light Guide
  • Excite exacte bulb
  • 12V halogen bulb

Troubleshooting

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

  • Turn off NIS-Elements
  • Turn off the camera
  • Wait few seconds
  • Turn the camera back on
  • Wait until the camera has fully 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"