Nikon TE2000-E inverted microscope

Light sources

Halogen lamp for transmitted light
X-Cite exacte for fluorescence

Emission (nm)	Nominal power (mW)

Objectives

4x/0.1 Air
10x/0.30 Ph1 Air
20x/0.45 Ph1 Air
40x/0.95 Air
60x/1.4 Ph3 Oil
100x/1.45 Oil

Position	Name	Brand	Full name	Identifier	Magnification	Numerical aperture	Immersion	Working distance (mm)	Transmittance (% [nm])	Applications	Cover glass thickness (mm)
1	4x/0.10 Air	Nikon	4x/0.1 Plan	MRL00042	4x	0.1	Air	30	>80% [400-900]	BF, Fluo	-
2	10x/0.30 Ph1 Aur	Nikon	10x/0.30 Plan Fluor Ph1 DL	MRH20101 MRH20105	10x	0.3	Air	15.2	>80% [440-700]	BF, PhC, Fluo	1.2 !
3	20x/0.45 Ph1 Air	Nikon	20x/0.45 Plan Fluor ELWD Ph1 ADL	MRH48230 MRH48220	20x	0.45	Air	7.4	>80% [350-870]	BF, PhC, Fluo	0-2
4	40x/0.95 Air	Nikon	40x/0.95 Plan Apo DIC M/N2	MRD00400 MRD70470	40x	0.95	Air	0.14	>80% [440-760]	BF, Fluo	0.11-0.23
5	60x/1.4 Ph3 Oil	Nikon	60x/1.4 Plan Apo Ph3 DM	MRD31600	60x	1.4	Oil	0.21		BF, PhC, Fluo	0.17
6	100x/1.45 Oil	Nikon	100x/1.45 Plan Apo Lambda OFN25 DIC N2	MRD01905 MRD71970	100x	1.45	Oil	0.13	>80% [460-720]	BF, Fluo	0.17
-	40x/0.60 Ph2 Air	Nikon	Plan Fluor 40x/0.60 Ph2 DIC M ELWD 0-2	MRH48430	40x	0.6	Air	2.7-3.7	>80% [350-850]	BF, PhC, Fluo	0-2
-	40x/0.60 Air	Nikon	S Plan Fluor 40x/0.6 ELWD DIC N1	MRH08430	40x	0.6	Air	2.8-3.6	>80% [350-850]	BF, Fluo	0-2
-	40x/0.90 Air	Nikon	S Fluor 40x/0.90 DIC M/N2	NA	40x	0.9	Air	0.3	NA	BF, Fluo	0.11-0.23
-	60x/0.70 Air	Nikon	Plan Fluor 60x/0.70 ELWD DIC M/N1	MRH08630	40x	0.7	Air	1.5-2.1	>80% [380-880]	BF, Fluo	0.5-1.5
-	60xA/1.40 Oil	Nikon	Plan Apo 60xA/1.40 Oil DIC H	MRD71670	60x	1.4	Oil	0.21	>80% [480-700]	BF, Fluo	0.17
-	60x/1.40 Oil	Nikon	Plan Apo VC 60x/1.40 Oil DIC N2	MRD71670	60x	1.4	Oil	0.13	>80% [480-700]	BF, Fluo	0.17
-	100x/0.85 Air	Nikon	L Plan 100x/0.85 ODN 25	MUE35900	100x	0.85	Air	0.85			0-0.7
-	100x/1.40 Oil	Nikon	Plan Apo 100x/1.4 Oil DIC H	MRD71970	100x	1.4	Oil	0.13	>80% [480-720]	BF, Fluo	0.17

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

Filters

Dichroic DAPI\FITC\TRITC
Dichroic CFP\YFP\Cy5
Dichroic CFP\YFP\mCherry
DAPI
Cy3n
Cy5.5

Also available

CFP
FITC
YFP
Cy3.5
mCherry
Cy5

Filter cubes

Position	Name	Brand	Identifier	Excitation	Dichroic	Emission	Comments
1	Dichroic DAPI\FITC\TRITC	Chroma	86013 v2		86013bs		To be used in combination with excitation and emission filters
2	Dichroic CFP\YFP\Cy5	Chroma	86008 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
4	DAPI	Chroma	31000 v2	AT350/50x	400dclp	D460/50m
5	Cy3n	Chroma	SP102 v1	HQ546/11x	Q557lp	HQ567/15m
6	Cy5.5	Chroma	41022	HQ665/45x	Q695lp	HQ725/50m
-	eCFP	Chroma	49001 ET	ET436/20x	T455lp	ET480/40m
-	cGFP	Chroma	31044 v2	D436/20x	455dclp	D480/40m
-	FITC	Chroma	41001	HQ480/40x	Q505lp	HQ535/50m
-	YFP	Chroma	49003 ET	ET500/20x	T515lp	ET535/30m
-	Cy3.5	Chroma	SP103 v1	HQ581/10x	Q593lp	HQ617/40m
-	mCherry	Chroma	49008 ET	ET560/40x	T585lpxr	ET630/75m
-	Cy5	Chroma	49006 ET	ET620/60x	T660lpxr	ET700/75m

Excitation filters

Position	Name	Brand	Identifier	Excitation	Dichroic	Emission
1	DAPI	Chroma	86013 v2 Exc 1	AT350/50x	86013bs	S457/17m
2	CFP	Chroma	86008 v2 Exc 1	S430/25x	86008bs 69008bs	S465/30m
3	FITC	Chroma	86013 v2 Exc 2	S484/15x	86013bs	S517/30m
4	YFP	Chroma	86008 v2 Exc 2	S510/20x	86008bs 69008bs	S550/50m
5	TRITC	Chroma	86013 v2 Exc 3	S555/25x	86013bs	S605/40m
6	mCherry	Chroma	89006 ET Exc 3	ET572/35x	69008bs	ET632/60m
7	Cy5	Chroma	86008 v2 Exc 3	S622/36x	86008bs	S700/75m
8	Empty

Emission filters

Position	Name	Brand	Identifier	Excitation	Dichroic	Emission
2	CFP	Chroma	86008 v2 Em 1	S430/25x	86008bs 69008bs	S465/30m
3	FITC	Chroma	86013 v2 Em 2	S484/15x	86013bs	S517/30m
4	YFP	Semrock	FF01-550/49-26	S510/20x	86008bs 69008bs	F01-550/49-25
5	TRITC	Chroma	86013 v2 Em 3	S555/25x	86013bs	S605/40m
6	mCherry	Semrock	FF01-641/75-25	ET572/35x	69008bs	FF01-641/75-25
7	Cy5	Chroma	86008 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

If not already done, turn on the computer (#1) and use your UdeM credentials to log in to Windows
Remove the cover from the microscope
Turn on the X-Cite exacte light source (#2)
Turn on the microscope power bar (#3)
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.
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.

If open, close NIS-Elements and wait until it is completely closed
On the Desktop open the folder Softwares
Open NIS Settings Utility
Click on the Import tab
Click on Browse
Navigate to your Documents
Select the file NIS Settings for Nikon TE2000E.bin
Click Select
Select all items
Click Import
Click OK
Close NIS Settings Utility
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.

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.

If not done already, press Escape to lower the objective to the lowest position
Place the test slide on the microscope stage with the coverslip toward the objective
Always use the test slide to perform the first focus.
If necessary, move the stage so that the sample is centered on the objective
In NIS-Elements, select the optical configuration BF vis or the desired fluorescence (DAPI vis, ...) to activate the configuration
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.
In NIS-Elements, select the optical configuration Off to turn off the illumination

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

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

After performing the first focus, in NIS-Elements:

In NIS, click Escape, to lower the objectives
Click 60x Oil or 100x Oil to select the desired objective.
The 100x objective has the greatest resolution (NA 1.45 vs 1.4)
Place a single drop of oil on the objective
In NIS-Elements, click again Escape to return the objective to its original position
Select the optical configuration BF vis or the desired fluorescence (DAPI vis, ...) to activate the configuration
Adjust the focus with the precision dial while looking through the eyepieces until the image is perfectly sharp
Select the optical configuration Off to turn off the illumination
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).

In any case, do not store your files on the C: drive.

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

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

Microscopie > Nikon TE2000-E (en) > Hamamatsu ORCA Flash V2 Spectral Response.png

Hamamatsu_Orca Flash 4.0_V2_C11440-22CU_Instruction Manual.pdf

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.