Thresholding for tiff stack looks at entire stack instead of individual tiff image

Question

I have a tiff stack (b/w) and when I open individual images from the stack in imageJ, the contrast is perfect, however, when i open the entire stack (image sequence of moving things), the contrast is terrible for the end slides (where fluorescence has of course, bleached out). IS there a way in either Matlab or imageJ to ensure that the contrast is determined not by the entire stack, but by the individual images in the stack (so that this change in brightness is no longer there).

Let me know how i can explain better. It's a really simple problem, but I don't know how easy it is to understand without seeing.

thanks

Answer 1

To correct a fluorescence signal bleaching over time, consider using the bleach corrector plugin for ImageJ. When thresholding a stack in ImageJ you can calculate the threshold separately for each slice:

• Image > Adjust > Threshold...

• leave Stack histogram unchecked to get a preview for the threshold calculated based on the current slice
• click on Apply

• in the dialog, choose Calculate threshold for each image to apply the chosen thresholding method to each stack slice separately.

In order to get the macro source code for this procedure, start the ImageJ macro recorder via Plugins > Macros > Record... before starting.