I often get asked how to uses Thresholds to measure things in Image J.
Below are some of the Basic Steps for using Thresholds:
- Open your image and duplicate it (Image>Duplicate)
- On the duplicate go to Image>Adjust>Threshold
- Play with the sliders until all of your cells are red.
- Click ‘Apply’
- You should now have a ‘binary’ black and white image
- Now go to menu Process>Binary and select ‘fill holes’
- You may also want to select erode, dilate, open or close to optimise the binary image so that you have nice solid filling of your cells.
- Now go to menu Analyse>Set Measurements. Select all the things you want to measure.
- Critical steps: make sure that you select your original image (not the binary) in the ‘Redirect to:’ pull down Menu
- Also make sure the ‘Limit to threshold’ checkbox is ticked and also tick the ‘Add to overlay’ and ‘Display label’.
- Click ok to close the ‘Set Measurements’ box.
- Now go to Analyse>Analyse Particles
- Here you will need to play around with the size and circularity settings (bit of trial and error) in order to get accurate identification of your cells or ROIs. I suggest making duplicates before you start so that you can quickly try different things to see which one works best.
- Make sure you have the Display results tick box selected.
- Once you click ok you should have a the measurements box appear with all your measurements for each cell.
- You can copy and paste these into Excel or what ever program you like to use.
- Go get a coffee and cake you deserve it!