Cell cluster invasion analysis using ImageJ

ImageJ:

1. Open ROI Manager (Analyze -> Tools -> ROI manager).

2. Open images with cell clusters.

3. Select freehand selection tool.

4. Trace cell cluster.

5. Click “Add” in ROI manager, or hit “t” on keyboard.

6. Close image.

7. Trace cell cluster in next image, Add in ROI manager.

8. Repeat for all images.

9. Select all ROIs (select first image in list, hold down shift and select last image) and click “Save” in ROI manager. Will save as .zip if there are many ROIs (leave unzipped!).

10. To get measurements, click “Measure” in ROI Manager.

11. Copy to excel sheet.

12. To place all cell cluster in same image (like examples above), open new image:

Type: 16-bit

Fill With: White

Width: 3072 pixels

Height: 2048 pixels

Slices: 1

13. If not still in ROI Manager, load images by clicking “Open” in ROI Manager and selecting .zip file with all ROIs.

14. Select all ROIs and click “Show All” in ROI manager.

15. Make sure freehand selection tool is selected. In ROI manager, click on first ROI, place cursor in center of cell cluster and move to desired position. Click “Update” in ROI Manager so ROI stays in new position.

16. Repeat for each cell cluster, leaving space in bottom left corner for scale bar.

17. Save new ROI positions by clicking “Save” in ROI Manager.

18. Fill images: in ROI Manager, under “More,” click “Fill.”

19. Save image as tiff.

Add scale bar in Photoshop:

20. To add scale bar, open tiff with merged cell clusters.

21. Image -> Analysis -> Select Measurement Scale -> Chaudhuri 63x (7 pixels = 100 pixels) (for 63x objective ONLY, create as new Custom measurement scale if not already made).

22. Image -> Analysis -> Place Scale Marker

Length: 100 (for 100 µm scale bar)

    Uncheck “Display Text”

        Color: Black

23. Save Image.