I have acquired some immunofluorescence images of a HeLa cell line stained against the processing body (P-body) marker DCP1A. As I have acquired the images at a high magnification (100x) all of the P-bodies are not in focus at a single z-plane. I have therefore acquired z-stacks during image acquisition to capture as many P-bodies as possible in focus. My goal is to quantify the number, pixel intensity and area of P-bodies.
My problem is that when I make a maximum intensity projection of my image stacks in ImageJ, out-of-focus light creates a halo effect around P-bodies in the final image. The halo subtly increases the size of the segmented P-body area when thresholding (see images below).
Maximum intensity projection:
Including fewer slices in the projection would help, but result in some P-bodies not entering focus and therefore not be detected in the segmentation step. Is there a method to get rid of or completely avoid the out-of-focus halo when doing z-stack projections? I am not a fan of the average intensity projection as it affects the pixel intensity quite profoundly. Any ideas?