Hi

I have to analyze microscopy images and I was not quite sure how. Perhaps you can advise me on possible analyzes or recommend me some bibliography.

My system consists of images obtained from confocal microscopy of cells infected with different pathogens. In my images I can see actin structures, which are formed due to the presence of bacteria (pedestals with EPEC, ruffles with Salmonella, and actin tails with Listeria). In these structures my protein of interest is also visualized. My hypothesis is that my protein plays a role in the formation of these structures through interaction with actin. Although my protein of interest is found throughout the cell, I would like to be able to show that it accumulates in the actin structures formed by these pathogens. Initially, I had thought about doing a co-localization analysis, but since my protein is found throughout the cell, I think the analysis may be confusing or uninformative. On the other hand, I considered evaluating the intensity profile, however I am not sure if it is possible to do a statistical analysis from the graph. Also, I would like to be able to carry out this analysis along the z axis.

I share with you some representative images. In EPEC and Salmonella, the bacteria express mCherry, actin is tagged with AlexaFluor647 phalloidin and my protein tagged with AlexaFluor488. In Listeria, the bacteria express GFP, actin is labeled with Rhodamine phalloidin and my protein is labeled with AlexaFluor 647 (in addition, the Ezrin protein is labeled with AlexaFluor488). The Cell image shows an uninfected cell, tagged with AlexaFluor647 phalloidin and my protein tagged with AlexaFluor488.

I would greatly appreciate any advice you can give me.

Thank you very much

Greetings