1. Turn on the computer (#1)
2. Remove the cover from the microscope
3. Turn on the microscope power bar (#2)

4. If incubation is required, turn on the Okolab incubation module (#3A) and the Lauda water-bath (#3B) and open the CO₂ (#3C) and N₂ (#3D) tanks

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   Make sure the humidifier and the water bath are clean and properly filled with distilled water.

   
   

5. Log in Windows using your UdM credentials
6. Start-up NIS-Elements

1. Save your data
2. Close NIS-Elements
3. Transfer your data to the D: drive (Data Storage) or to your external drive and delete it from the local C: drive
4. Turn off the computer
5. If oil objectives were used, clean it with lens cleaner and lens paper (not Kimwipes)
6. If incubation was used, turn off the Okolab incubation module (#3A) and Lauda water-bath (#3B) and close the CO₂ (#3C) and N₂ (#3D) tanks
7. Wait until the SpectaX has cooled down and turn off the microscope power bar (#2)
8. Cover the microscope

• 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).

The first time you use the instrument, 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.

1. If open, close NIS-Elements and wait until it is completely closed (up to 30 seconds)
2. On your Desktop open the Softwares folder
3. Open NIS Settings Utility
4. Click on the Import tab
5. Click on Browse
6. Navigate to your Desktop
7. Select the file Nikon-Ti2 Settings.bin
8. Click Select
9. Select all items
10. Click Import
11. Click OK
12. Close the NIS Settings
13. You can now reopen NIS-Elements

• Bring long Rj45 cable to connect Acquisition and Processing workstations
• Check condenser filters if labels match
• Check why 20x/0.75 DIC objective has no DIC prism
• Condenser DIC prism is DIC N1 while objective requires N2
• Check Liquid light guide transmittance

• 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 Illumination Report_Nikon Ti2_2021-11-05.pdf
• 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 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

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

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

• Remove distilled water from the humidifier

• 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

• Wipe any liquid with a tissue
• Reconnect the yellow tube to the incubation chamber 

• 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
• For long timelapse, be aware of photo-toxicity

You may use a trick to warmup the incubation chamber faster. Using this method. you should reach a stable temperature within 30 minutes.

• 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. 

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