77074656 Excitation filters are in the Lumencor SpectraX light source
Detector
Hamamatsu ORCA Flash V2 C11440-22CU CMOS Monochrome Camera 2048 x 2048 pixels, 16-bit, 30 images/s at full frame
Turn on the computer (#1)
Remove the cover from the microscope
Turn on the microscope power bar (#2)
If incubation is required, turn on the Okolab incubation module (#3A), the Lauda water bath (#3B) and open the CO2(#3C) and N2(#3D) tanks
Make sure the humidifier and the water bath are clean and properly filled with distilled water.
Use your UdeM credentials to log in to Windows
Start the software NIS-Elements
The first time you use the instrument, you need to import the microscope settings into the software. To do this follow the instructions First use protocol.
Save your data
Close NIS-Elementssoftware
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
If oil objectives were used, clean it with lens cleaner and lens paper (not Kimwipes)
If incubation was used, turn off the Okolab incubation module (#3A),Lauda water bath (#3B) and close the CO2(#3C) and N2(#3D) tanks
Wait until the SpectaX has cooled down and turn off the microscope power bar (#2)
Cover the microscope
Important Reminders
Collect your samples, especially those in the microscope
Leave the microscope and workspace clean
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).
Important
In any case, do not store your files on the C: drive.
When using the microscope for the first time, you need to import the microscope settings into the software. You will usually do this during the training session. This procedure can also be performed if something is not working properly and if you want to reset the software to its original settings.
This process will delete all experiment protocols and reset the software to the original settings for this specific microscope.
If open, close NIS-Elements and wait until it is completely closed (up to 30 seconds)
On your Desktop open the Softwares folder
Open NIS Settings Utility
Click on the Importtab
Click on Browse
Navigate to your Desktop
Select the file Nikon-Ti2 Settings.bin
Click Select
Select all items
Click Import
Click OK
Close the NIS Settings
You can now reopen NIS-Elements
The following schematics depict the light path for transmitted (bright-field and Phase Contrast) and reflected (fluorescence) lights.
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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 wikipedia
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
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
20x/0.5 Air Ph1 WD 2.1
60x/1.4 Oil DIC WD 0.13
100x/1.45 Oil Ph3 WD 0.13
100x/1.45 Oil DIC WD 0.13
4x/0.2 Air WD 20
20x/0.75 Air DIC WD 1.0
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 3cm 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 CMOS Monochrome Camera 2048 x 2048 pixels, 16-bit, 30fps at full resolution Serial 101081
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
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
Consumables
CO2 Tank
N2 Tank
Liquid Light Guide
Troubleshooting
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 unlabeled 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 warmup 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
Immerge 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
