I am an undergrad just getting started with ImageJ and I am unsure of how to quantify cell fluorescence on some images I have taken.
For context: I stained cells with two fluorophores, DAPI and PerCP. I currently have .lif image files of these slides, where each image file contains three different, adjacent images: one taken with visible light, one at DAPI emission wavelength, and one at PerCP emission wavelength. I have installed the BioFormat plugin for ImageJ so that the software is able to open the .lif image files.
I would like to use the DAPI image to define areas of interest on the slide (i.e. I am only interested in analyzing the PerCP fluorescence of cells that displayed DAPI staining). Once I have used the DAPI image to select areas of interest, I would like to quantify the level of PerCP staining in those areas of interest, perhaps by converting the PerCP image to greyscale and measuring the brightness.
I have to admit I really have no idea how to start. I’ve googled around a bit but have not had success at accomplishing this task, I think I just lack the software familiarity needed to engage in trail-and-error intelligently, so I’ve just been floundering around not knowing what’s going on.
Does anyone have any tips on how to:
- select only regions that exhibited DAPI fluorescence for further fluorescence quantification analysis?
- convert PerCP fluorescence to greyscale and measure the brightness in the areas of interest?
Thanks so much!