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
Click to enlarge
Click to enlarge
Description
Light sources
LED lamp for transmitted light
CoolLed pE340-fura
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
Lumencor Spectra
Channel
Source
Filter
Bandwith
Fluorophore
Nominal 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, BFP
301
Cyan
473
473/28
[459-487]
GFP, FITC, Alexa Fluor 488, Oregon Green, SYBR Green
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
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.
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
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.
On the microscope, gentlypush 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
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
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
Gentlyreturn 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
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:
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 !
First perform the initial focusing with the safest objective before selecting a higher-magnification objective
After completing the initial focus:
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).
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 EscapeZ 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!
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)
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!
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.
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
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
Neutral Density ND
Empty
Empty
Empty
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
DAPI Cube Semrock Brightline 96370 M352024 1 No excitation filter
GFP Cube Semrock Brightline 96372 M380163-17 GFP-4050B Ex 466/40 Em 525/50 Di 495
Cy3 Cube Semrock Brightline 96374 M380162-10 LED-TRITC-A Ex 554/23 Em 609/54 Di 573
Cy5 Cube Semrock Brightline 96376 M351081 23
Fura77074017 79001 ET Fura2 C189116 (smaller cube not 25mm) No Excitation Filter
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
