Within the software IN Cell Analyzer:

1. Click Eject to open the access door
2. Insert your sample in the space provided for this purpose, respecting the orientation indicated

3. Click Load to close the access doors

   First use

   During the training you will follow the First use protocol

   Important

   For use with the 60x objective it is absolutely necessary to use a multi-well plate with a glass bottom. The glass thickness should be 0.17mm. We recommend the following plates:

1. Collect your samples
2. Click Load to close the access door
3. Close the INCell Analyzer software
4. Transfer your data to disk D: (Data Storage) or your external hard drive and delete it from local disk C:
5. Turn off the computer

• Files can be saved temporarily (during acquisition) to local disk C: (desktop)
• At the end of each session, copy your data to your external drive and delete it from local drive C:
• You can store your files on disk D: (Data Storage). If using this disk, please create one folder per laboratory using the principal investigator's last name. Inside, create a folder per user (First Name_Last Name).

When using for the first time, it is necessary to define the type of plate used.

Create a new plate

In the INCell Analyzer software:

1. Select from the menu Application>Plate\Slide Manager...
2. Click on the New Plate\Slide icon
3. Select the number of wells in your plate (6, 24, 96 etc...)
4. Adjust the following settings:
   1. Name: Your-Name_Your-Plate
   2. Plate height, bottom thickness and well volume usually provided in the product technical sheet by the manufacturer
   3. The material used plastic or glass
   4. If necessary adjust the number of rows and columns as well as the shape of the well (round or rectangular)
5. Click OK
6. Close the Plate/Slide Manager window

Measure the bottom height and thickness of the plate bottom

In the INCell Analyzer software:

1. Click on the Dashboard
2. In Objective Lens select the 10x objective
3. On the plate plan, click on the center of a well containing a sample to center this well on the objective
4. In the Dashboard menu select Plate/Slide
5. Click Verify LAF
6. If the 2 detected peaks (blue vertical lines) correspond to the measured peaks (black curve)
   1. Click Apply Measured Parameters
   2. Click OK
   3. Click Yes
7. If the 2 detected peaks (blue vertical lines) do not correspond to the measured peaks (black curve)
   1. Change the bottom thickness value and repeat the operation
8. Close the Laser Autofocus Plate/Slide Verification window

Measure A1 well offset

In the INCell Analyzer software:

1. if necessary, click on the Dashboard > Objective Lens select the 10x objective
2. In the channel list, click the + button and add a transmitted light channel (brightfield)
3. To the right of the plate layout, click the Setup Preview Imaging button
4. Draw a rectangle around well A1
5. Click on preview (on the acquisition panel at the top right of the screen)
6. Wait for the preview to be generated
7. On the plate map, click on the real center of well A1 to center this well on the objective

8. In the Dashboard menu select Plate/Slide

9. Click Edit Plate then Define Upper Left Well

10. Click Yes

11. Check that the yellow circle matches the edges of the well

12. Click Yes

Compute the inter-well distance

• Repeat the offset measurement steps for the bottom right well

• The inter-well distance will then be automatically calculated

• Added to wiki

Usually, this microscope displays a green light when operational and an orange light when in use. A red light is on means the microscope is not functional. In this case please contact the platform manager.

Can I use this microscope to look at cell in a dish?

• No. This a microscope designed to acquire images of specimen in multi-well plates
• You may also image specimen mounted between a slide and a 0.17mm thick coverslip