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

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id	Description
title	Description

Description

Light sources

LED lamp for transmitted light

CoolLed pE340-fura

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title	Complete specifications

Channel	Source	Filter	Fluorophore	Nominal Power (mW)	Measured Power (mW)
1	340nm	BP340/20	Fura	?	TBD
2	380nm	BP380/20	Fura	?	TBD
3	435-645nm	-		?	TBD

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name	CoolLED_pE-340fura_User-Manual.pdf
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name	CoolLED_pE-340fura_Quick Start Guidepdf.pdf
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name	CoolLED_pE-340fura_Troubleshooting Guide.pdf
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name	CoolLED_pE-340fura_Diagram.pdf
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name	CoolLED_pE-340fura_Datasheet.pdf
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Lumencor Spectra

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title	Complete specifications

Channel	Source	Filter	Bandwith	Fluorophore	Nominal Power (mW)
Violet	390	390/22	[380-400]	DAPI, Hoechst, coumarins	236
Blue	437	437/26	[424-450]	CFP, Alexa Fluor 430, Pacific Blue, BFP	301
Cyan	473	473/28	[459-487]	GFP, FITC, Alexa Fluor 488, Oregon Green, SYBR Green	179
Teal	512	512/18	[503-521]	GFP (secondaire), YFP (partiellement), Alexa Fluor 514, SYTO dyes	60.6
Green/Yellow	542	542/30	[527-557]	Alexa Fluor 546, Cy3, TRITC	355
Green/Yellow	575	575/30	[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

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name	Lumencor_Spectra_User-Manual.pdf
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name	Lumencor_Specra_Certificate of Conformance.pdf
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name	Lumencor_Spectra X_Manual.pdf
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Objectives

4x/0.2 Air
10x/0.45 Air
20x/0.75 Air
40x/0.75 Air
60x/1.4 Oil

-

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title	Complete specifications

Position	Name	Brand	Full name	Identifier	Working distance (mm)	Transmittance (% [nm])	Techniques	Cover glass thickness (mm)
1	4x/0.2 Air	Nikon	4x/0.2 Air CFI Plan Apochromat Lambda	MRD00045	20.0		BF, Fluo	0.17
2	10x/0.45 Air	Nikon	10x/0.45 Air CFI Plan Apochromat Lambda	MRD00105	4.0		BF, DIC N1, Fluo	0.17
3	20x/0.75 Air	Nikon	20x/0.75 Air CFI Plan Apochromat Lambda	MRD00205	1.0		BF, DIC N2, Fluo	0.17
4	40x/0.75 Air	Nikon	40x/0.75 Air Plan Fluor	MRH00400	0.72			0.17
5	60x/1.4 Oil	Nikon	60x/1.4 Oil CFI Plan Apochromat Lambda	MRD01605	0.13		BF, DIC N2, Fluo	0.17
6	-	-	-	-	-	-	-	-

Filters

DAPI
GFP
Cy3
Cy5
Fura
-

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title	Complete specifications

Position	Name	Brand	ID	Excitation Filter	Dichroic mirror	Emission Filter	Comments
1	DAPI	Semrock	96370 M352024	-	409LP	447/60	Semrock Brightline 96370 M352024 DAPI-3060A No excitation filter
2	GFP	Semrock	GFP-4050B	466/40x	495LP	525/50m	Semrock Brightline 96372 M380163-17 GFP-4050B
3	Cy3	Semrock	LED-TRITC-A	554/23	573LP	609/54	Semrock Brightline 96374 M380162-10 LED-TRITC-A
4	Cy5	Semrock	Cy5-5070A	618/50	652LP	698/70	Semrock Brightline 96376 M351081 23 Cy5-5070A
5	Fura	Chroma	79001-ET-Fura-2	-	400LP	510/80	Chroma Fura 77074017 79001-ET-Fura2 C189116 Smaller cube (not 32mm). No Excitation Filter.
6	-	-	-	-	-	-	-

Detector

Photometrics Prime 95B 25mm

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title	Complete 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 resolution	30 images/s
Max QE	95 %
Readout noise	1.6 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

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name	Photometrics_Prime-95B_Datasheet.pdf
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name	Photometrics_Prime-95B_Manual.pdf
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title	User Guide

User Guide

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title	Start-up

If not already done, turn on the computer (#1) and log in to Windows using your UdeM credentials
Remove the dust cover from the microscope
Turn on the microscope power bar (#2) on the desk between the microscope and the computer
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
Start NIS-Elements

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title	First 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. If you are not sure, ask for support.

If NIS-Elements is open, close it and wait until it has completely shut down (this may take up to 30 seconds)
On the Desktop, open the Softwares folder
Open NIS Settings Utility
Click on the Import tab
Click on Browse
Navigate to your C:\Users\Public\Documents
Select the file Nikon_Ti2-Fura_NIS Settings.bin
Click Select
Select all items
Click Import
Click OK
Close the NIS Settings Utility
You can now reopen NIS-Elements

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title	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.

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title	Initial Focus

On the microscope, gently push the transmitted light arm backward
In NIS-Elements, click Escape Z to move the objectives to a safe position

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

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
If necessary, use the joystick to move the stage so the sample is centered under the objective
Gently return the transmitted light arm to the vertical position
In NIS-Elements, click Escape Z again to return the objectives to their normal position
Click on the desired optical configuration (BF, DAPI, GFP, etc.)
Click Live (») to activate the light and display the camera image
Adjust the light intensity and exposure time to obtain a well-exposed image
Adjust the focus until the image is perfectly sharp.
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

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title	Create 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:

In NIS Elements, click on the desired optical configuration (BF, DAPI, GFP, etc.)
Click Live (») to activate the illumination and display the camera image on the screen
Adjust the light intensity and exposure time to obtain a properly exposed image
Using the Joystick navigate to the top left corner of your sample
In the the XYZ overview click + to add a point and save this position
Using the Joystick navigate to the bottom right corner of your sample
In the the XYZ overview click + to add a point and save this position
In the XYZ Overview right click and under Large Image Area select Define Area
Draw a rectangle around the two points
Right click again on the created region of interest and select scan preview
Wait until the preview acquisition is completed

You can now directly click on the overview to navigate your sample !

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title	Secondary focus

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

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title	Focusing with air objectives

After completing the initial focus:

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).
Click on the desired optical configuration (BF, DAPI, GFP, etc.)
Click Live (») to activate the illumination and display the camera image on the screen
Adjust the light intensity and exposure time to obtain a properly exposed image
Adjust the focus using the precision knob until the image is perfectly sharp
Click Stop to stop the Live mode or click on the optical configuration Off to turn off the illumination
Click Escape Z to move the objectives to a safe position
On the microscope, you can remove the test slide and install your sample
In NIS-Elements, click Escape Z once again to return the objectives to their normal position.

Your sample is now ready for acquisition!

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title	Focusing with oil objectives

After completing the initial focusing:

In NIS-Elements, click Escape Z to move the objectives to a safe position
Click on the desired immersion objective (60x)
Info
The 60x is the best immersion lens because it has the highest numerical aperture (1.4).

Remove the test slide
Place a single drop of oil on the objective
Place your sample with the coverslip facing toward the objective
Click Escape Z again to return the objectives to their normal position
Click on the desired optical configuration (BF, DAPI, GFP, etc.)
Click Live (») to activate the illumination and display the camera image on the screen
Adjust the light intensity and exposure time to obtain a properly exposed image
Adjust the focus using the precision knob until the image is perfectly sharp
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!

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title	Storage management

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).

Note
In any case, your files should be removed from the C: drive.

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title	Shutdown

Save your data
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
If used, clean oil objectives with lens cleaner and paper
Select the lowest magnification objective and click Escape Z to place the objectives in a safe position
Wait until the SpectaX fan is off and then turn off the microscope power bar (#2)
Turn off the computer
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

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title	Log

Log

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title	To do

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

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title	2025-10-09 Installation Spectra

Installation Spectra
Installation LAPP

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title	2025-03-10 Insallation Desmarais 2236

Complete installation
Complete cleaning

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title	Technical Datasheet

Technical Datasheet