This guide can be used to manually count cells on whole tissue sections scanned using the slide scanners. It is best used when the cells to be counted are in small populatione as in the example provided.

This method offers several advantages:

• It is faster and more reliable than having to count in ImageJ or Photoshop.
• The counts can be saved so they can be reviewed or audited at a later date.
• Co-expressing cells are detected by the software (avoids counting with 3 cell markers instead of 2)
• The whole slide is visible so other features or cell populations can be counted or measured at a later date.
• Image acquisition using the slide scanners is autonomous, up to 80 slides at a time, so only post-imaging analysis is necessary

1. Open the .ims file of the slide into Imaris

2. Create a new spots layer by clicking on the top spots icon and choose skip automatic creation, edit manually

3. Under the edit / pencil tab you will be able to place spots manually – using these spots you can count the cells. To place spots you will need to be in select mode instead of navigate mode. To quickly change from navigate to select press the escape key. You will now be able to place a spot anywhere on the image by holding down the shift key and left clicking.

4. The spots may difficult to see, so under the main spots tab (circled) enter a new spot radius – 8 is usually a good size for counting cells.

5. If you have more than one marker you can create additional spot layers. If you are counting a second marker and don’t want to be distracted by the previous count you can toggle its visibility using the check box.

6. Once you have finished counting you can get the total cell count by clicking on the statistics tab. These values can be easily exported to excel using the buttons below this table.

7. If you are counting using several cell markers and also need to count the co-labelled cells it is not necessary to individually recount these cells. Once you have finished counting the two separate markers go the the advanced tab of one of the spot layers and choose “colocalise spots”.

8. Choose the two spot layers you wish to colocalise (e.g. grey and pink cells).

9. Type in the distance (in µm) to detect colocalised cells. Usually somewhere between 5-10µm will give accurate results.

10. Imaris will then create a new set of spots which show all the co-labelled cells. Again, the total count for the cells can be found in the statistics tab.

11. When you have finished counting or if you need to stop counting and don’t want to loose the work you have completed it is possible to save the spots withing having to resave the huge image file you working on. To do this, open a new image into imaris (preferably a small .tif file) – this will keep all the spot counts and locations but remove the slide scanner image. Save this file. Later, to continue working or if you need to check your counts, open this file in imaris, then open the original slide scanner image – all the spots should be located as you orignally placed them.