I am trying to segment foci with microbeJ. The foci are observable by eye, but have varying intensities. The background intensities are also variable. Some cells fluoresce entirely, but do not have localized expression - meaning that there is no foci (see image attached). However MicrobeJ is still grabbing the maxima and calling it a foci. I would like to limit what MicrobeJ calls to sharp changes in intensity and small localized fluorescence (but in a way that is lenient to varying intensities across multiple cells). I cannot figure out how to adjust the microbeJ settings to achieve this. The example images attached are two cells: one with zero foci but with background expression, and one with 2 foci. Any help would be great.
You could potentially threshold using a local threshold and create a mask. Then analyse particles.