Measure pixel intensities in images of the cells obtained by epifluorescence microscope

I am a beginner in fluorescence microscopy. Can some give me an advice? I have cells fixed on the slides and stored in -20°C (they are from different patient groups). I would like to stain them fluorescently. Will it be possible to use these cells for measuring of pixel intensities, if I will capture pictures by epifluorescence microscope? Need I for these purposes multidimensional (2D, 3D) pictures?

Dear @Eva_Novosadova,

welcome to the forum! :slight_smile:

I think it’s close to impossible to provide a general answer to that question. If you can acquire images (doesn’t matter if it’s single planes or multiple slices), you can measure pixel intensities. Those intensities might for example be close to zero depending on your imaging modalities. What I am trying to say is, that it heavily depends on the combination of your cells, the stain, and the microscope (i.e. camera, excitation system, etc…).

For an automated analysis of intensities you should try to optimize the signal-to-noise ratio of cells and background.

I would say, that heavily depends on the biological question you are trying to answer.


Dear Stefan, thank you for your answers. Our aim is to compare fluorescence stainig in two T lymphocyte populations and compare them between two groups of patients. We would like to take pictures of these cells and count pixel intensities. But I do not know if epifluorescence microscop will be suitable for this purposes.