Roger Gaudry Building, Room R-619 Advanced Microscope Tier 1 usage price Instrument awarded to Dr Paradis-Bleau the Canadian Foundation for Innovation (CFI #34495) in 2015
77074656 Excitation filters are in the Lumencor SpectraX light source
Detector
Hamamatsu ORCA Flash 4.0 V2 C11440-22CU
Camera
Hamamatsu ORCA Flash 4.0 V2
Sensor Type
CMOS
Sensor Category
Monochrome
Nb Pixels
4.2 M
Pixel Layout
2048 x 2048
Pixel size
6.5 um
Sensor size
13.312 mm x 13.312 mm
Sensor diameter
mm
Bit depth
16-bit
Speed at full resolution
30 images/s
Max QE
83 %
Readout noise
1.6 e⁻
Cooling
-10 C Air
Dark Current
0.06 e⁻/pixel/sec
Full well capacity
30 000 e-
Dynamic Range
1: 37 000
Interface
USB 3.0
Mount
C-mount
User Guide
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
if incubation is required, turn on Okolab incubation module (#3A), the Lauda water bath (#3B) and open the CO₂ (#3C) and N2 (#3D) cylinders near the door
Make sure the humidifier and water bath are properly filled with distilled water.
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-Oko_NIS Settings.bin
Click Select
Select all items
Click Import
Click OK
Close the NIS Settings Utility
You can now reopen NIS-Elements
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.
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, gently push the transmitted light arm backward.
In NIS-Elements, click Escape to move the objectives to a safe position.
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 parfocal, focusing with these objectives 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.
Gently return the transmitted light arm to the vertical position.
In NIS-Elements, click Escape 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 screen.
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 objective (20x PhC or 20x DIC)
The 20x DIC objective 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 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 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 to move the objectives to a safe position.
Click on the 63x, 100x DIC, or 100x PhC objective to select the desired lens.
The 100x DIC objective is the best oil immersion lens because it has the highest numerical aperture (1.45).
Place a single drop of oil on the objective.
Click Escape 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!
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 press escape to place the objectives in a safe position
If used, turn off the Okolab incubation module (#3A),Lauda water bath (#3B) and close the CO2(#3C) and N2(#3D) cylinders
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
Condenser DIC prism is DIC N1 while objective requires N2
Check why at 4x Condenser gives a black shadow on the righ side of the image
NIS Objective Z offset
Nitrogen Tank replacement Praxair 1066 106723504
CO2 Tank replacement Praxair 1013 106722901
Migrated to Windows 11
Fixed Stage insert bent
Cleaning all objective and filter cubes
Added XY Offset
Fixed incubator tubing misplaced
Added better attachment for tubing on the incubation chamber
Checked incubation chamber for obstruction
Fixed incubator tubing misplaced
Added better attachment for tubing on the incubation chamber
Checked incubation chamber for obstruction
Fixed condenser holder loose
20x/0.5 Ph1 dirty or damaged likely dirty with varnished
Added network cable between Acquisition and Processing workstations
Added shortcuts in Desktop\Documentation
Clean 60x objective (nail polish on it !)
Fix damaged stage insert
Stage insert tilt adjustment
Objective XY offset adjustments
Installed new objective 4x/0.2 Plan Apo Lambda
Updated configuration file
Computer maintenance
Imaging test slides
Re-install DCAM API drivers v22.2.6391
Okolab incubation complete maintenance
Disassemble Okolab stage insert
Disconnect the pipes
Disconnect the Lauda water-bath
Clean everything with 10% acetic acid (excepted the plastic pipes and washers)
Rinse with regular tap water 2 times
Rinse with distilled water once
Dry, reassemble and reconnect
Refill with the Lauda water-bath with 3L of milliQ water
Set the Okolab temperature Control Mode to Chamber
Set the Okolab temperature to 65°C
Let run for 1h. This will sterilize the water.
Check for any leakage
Set the Okolab temperature Control Mode back to Sample
Set the Okolab temperature back to 37°C
Cleaning objectives
Control for incubation chamber liquid intrusion
CO2 Calibration for Okolab: Offset of 1%
Control for illumination quality
See
Liquid light guide adjustment
20x/0.75 DIC objective was dirty and has been cleaned
Printed and displayed a Memo about available air and oil objectives
Adjustment of camera angle
Objective calibration
Objective XY offset
Updated NIS settings
Added light path schematics to Wiki
Okolab display a NAN message for the free thermal sensor
The probe is made of two wires that need to be in contact to measure the temperature properly
Turn off the Okolab module
Disconnect the free sensor probe
Cut out the damaged part
Carefully expose the two wires
Connect the two wires together
Added label on gas bottles
Added line mark on humidifier
512 GB SSD installed for OS
Windows 10 Installation
BIOS updated to v2.47
Technical Datasheet
Stand
Nikon Ti2-E inverted Serial 540156 System 170110-Sys-006287
Light sources
Transmitted LED light
ND32 filter
IR filter
Manual Polarizer
Lumencor SpectraX 6-NII-SE Serial 9409
Condenser
Motorized condenser Ti2-C-TC-E Serial 519097
Lens LWD NA 0.52
Filter turret 7 motorized positions
Empty
Empty
Ph1
Ph3
Shutter
Empty
DIC N1
Objectives
4x/0.2 Air MRD00045
20x/0.5 Air Ph1 MRH10201
20x/0.75 Air DIC MRD00205
60x/1.4 Oil DIC MRD01605
100x/1.45 Oil Ph3 MRD31905
100x/1.45 Oil DIC MRD01905
Stage
Motorized stage Ti2 SHU compatible Serial 127808
Remote control joystick Ti2-S-JS Serial 127976
Inserts
Multi-well plate Ti2-S-HW with tilt adjustment (no incubation)
Combo slide 3 cm dish Ti2-S-HU with tilt adjustment (no incubation)
Okolab H101-CellASIC Frame with perfusion ports
1 x 35mm petri dish 1x35-M + cover
2 Chamber Slide 2xGS-M+ cover
1 Multi-well for oil objectives MW-OIL
6-well plate 6MW+ cover
1 CellASIC + cover
Filters
DAPI Cube Ex 383-408 DAPI-U DM 425 BA 435-485
GFP Cube Semrock 96372 M349727 17
Cy5 Cube Semrock 96376 M351081 8
77074160 Custom Quad C182279 Polychroic and quad bandpass emitter for use with the following single bandpass filters: ET395/25x, ET470/24x, ET550/15x, ET640/30x
DIC Analyser Ti2-C-DICACL
C197767 7707\4656 CFP\YFP\mCherry XT
Detector
Hamamatsu ORCA Flash V2 C11440-22CU Serial 101081
Workstation
HP Z440 Workstation
I155431-CIB
Intel Xeon E5-1620 v4 @ 3.5GHz
RAM 32 GB DDR4-2400 1200 MHz ECC (4 x 8 GB)
OS 500 GB NVMe via PCIe to M2 Adapter 2 500 MB/s
4 TB HD Data Storage (2 x 2 TB spanned volume) 130 MB/s
Video Card nVidia GTX 1080 8GB GDDR5 dedicated memory
Monitor HP Z24i display 24' 1920x1200
Software NIS-Elements AR v5.02
Incubation
Okolab BoldLine Temperature unit Serial 284-1058 H101 T Unit BL
Okolab BoldLine CO2/O2 Unit 0-10/1-18 Serial 088-1102
Okolab OkoTouch Serial 118-224
Lauda water-bath Model Eco RE415 S LCK 4910 Serial LCK-4910-16-0006 Okolab 1322-1006 2017-05-30
This can happen when the liquid running through the chamber is not flowing properly.
Pause your experiment
Turn off the Okolab environment controller 3A and 3B
Disconnect the blue end of the connection pipe (at the junction with the spring shape objective warmer)
Place the open end into a dish to collect liquid
Turn the Lauda water bath back ON (3B)
The liquid should flow quite rapidly, if not proceed as follow
Turn OFF the Lauda water bath (3B)
Take the 20 mL syringe located inside the drawer labelled Tubing
Connect it to the open end of the blue pipe
Use the syringe to blow pressurized air into the pipes to clear it out
Remove the syringe (and store it back into the Tubing drawer)
Test if the liquid is now flowing properly by turning the Lauda water bath back ON (3B)
If not repeat the procedure
If so, turn OFF the Lauda water-bath (3B)
Reconnect the blue and green pipes together
Turn the Okolab environment controller 3A and 3B back ON
Be careful not to touch your sample when performing these actions as it may displace your current acquisition
How the Okolab chamber works
The liquid from the water bath enters the lead first via the red pipe
Then it goes through the lead circuitry to keep it warm
It exists via the unlabelled tube and enters the incubation chamber main body
It goes through the circuitry of the main body and exits via the green labelled tube
One must maintain a decent amount of liquid in the Lauda water bath to avoid bubble formation in the circuitry. Because the circuitry inside the top lead and the incubation chamber main body is thin it can get clogged. The procedure above can solve this issue.
This happens when the mixed-gas humidifier is overfilled. Bubbles created by the gas going through the humidifier can bring liquid into the gas feed line.
Turn off the Okolab module and the water bath
Remove your sample and store it properly
Dry the incubation chamber with a clean tissue
Carefully remove the cap of the humidifier glass bottle
Pay extra care when manipulating the humidifier bottle as it is made of glass and is very fragile
Remove distilled water from the humidifier
Humidifier shouldn't be more than 2/3rd filled
Close the humidifier by replacing the cap
Turn on the Okolab module and the water bath
This can happen during long experiments. The high humidity of the gas mixture condensates in the pipe between the humidifier and the incubation chamber.
Pause your experiment
Disconnect both ends of the yellow tube from the humidifier
Drain the tube from any liquid
Reconnect the yellow tube to the humidifier
You can use a syringe or gas pressure to blow out any liquid from the incubation chamber cover
Be careful not to touch your sample when performing this action
Wipe any liquid with a tissue
Reconnect the yellow tube to the incubation chamber
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
You may use a trick to warm-up the incubation chamber faster. Using this method. you should reach a stable temperature within 30 minutes.
Do NOT proceed with your sample but with a blank control (dish with distilled water for example)
Place your blank control in the incubation chamber
Immerse the tip of the sample temperature probe in the blank
Set the Okolab temperature Control Mode to Chamber (Settings>Temperature>Control Mode>Chamber>Save)
Set the Okolab temperature to 50°C (Home>Temperature>50°C>Set)
Check the temperature of the sample (Menu Magnifier>Sample Temperature). It should take between 10 to 15 minutes for the blank to reach 30°C.
The Lauda water bath is faster to warm up water than to cool it down. Make sure to anticipate and not pass beyond the desired temperature. If your desired temperature is 37°C, you can stop the procedure when the blank has reached 30°C.
When the blank has reach the desired temperature
Set the Okolab temperature back to 37°C (Home>Temperature>37°C>Set)
Set the Okolab temperature Control Mode to Sample (Settings>Temperature>Control Mode>Sample>Save)
Wait 10 to 20 minutes until the temperature stabilizes
Then you can safely replace the blank with your sample
