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 scan an overview 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
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
Fix camera adapter
Purchase 3mm adapter for Lapp
Purchase multiband pass cube for Spectra
Fix stage inserts
Complete installation
Complete cleaning
Stand
Nikon Ti2-E inverted Serial System
Light sources
Transmitted LED light
ND filter: TBD
CoolLED pE340-Fura Serial
Lumencor Spectra Serial Serial
Condenser
Manual condenser Serial
Lens LWD NA 0.52
Filter turret 7 manual positions
ND TBD
Empty
Empty
Empty
Empty
Objectives
4x/0.2 Air WD 20
10x
20x/0.75 Air DIC WD 1.0
40x
60x/1.4 Oil DIC WD 0.13
Stage
Motorized stage Encoded Serial
Remote control joystick Ti2-S-JS Serial
Inserts
Combo slide 3cm dish Ti2-S-HU with tilt adjustment (no incubation)
Filters
DAPI Cube TBD
GFP Cube TBD
Cy3 Cube TBD
Cy5 Cube TBD
Fura
Detector
Photometric Prime95B 25mm Serial A18C203010
Workstation
HP Z440 Workstation
Intel Xeon E5-1620 v4 @ 3.5GHz
RAM 32 GB DDR4 2400 MHz ECC (4 x 8 GB)
OS 500 GB SSD 530 MB/s
4 TB HD Data Storage (2 x 2 TB spanned volume) 130 MB/s
Video Card nVidia GTX 1080 8GB DDR5 dedicated memory
Monitor HP Z24i display 24' 1920x1200
Software NIS-Elements AR v5.02
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
