I am new to using CellProfiler and have limited experience using ImageJ and would greatly appreciate some advice on how to best perform the analysis for my experiment.
I have images from cells that were stained with MitoTracker GreenFM (for mito. mass) and MitoTracker Red Cmx Ros (for mito. membrane potential) and imaged in a 5x5 panel at 20x on a 12-well plate using an automated live-cell microscope (Lionheart). There is some overlap between the images as they were subsequently stitched into a single image (Unless the stitched images are used for the analysis, it may be necessary to take this into account so that the same cells aren’t measured twice). There are 4 treatment groups corresponding to each column of the plate (3 replicates per group).
Ultimately, I need to determine the fluorescence intensity of both channels so that I can calculate the mitochondrial membrane potential relative to mass.
The images are saved as individual tiffs for each channel (“GFP” and “Cy5”) and do not appear to contain any metadata beyond the file names. The regular expression for the files is “” so for example: “A1_1_GFP.tiff” and the stitched images are named like “A1_1_Stitched[GFP 469,685]” for example (Note: anything within the brackets will not seem to work for organizing images in the “NamesAndTypes” module on CellProfiler).
My goal is to design a pipeline in CellProfiler (or a script/macro in imageJ) to accomplish this task in an automated fashion. I would need to keep track of the matching images so that the ratios are calculated appropriately (ie. A1_1_GFP and A1_1_Cy5 are from the same field/location). So first of all, any advice as to how to set up my “NamesAndTypes” module in CellProfiler would be much appreciated.
Next, I would like to create an outline of each cell in the image, by using thresholding and/or generating a mask (I’m assuming using the “IdentifyPrimaryObjects” module in CellProfiler might accomplish this task) and then measuring the fluorescence intensity on both channels within the same outlined area (mask?). Even better if this can be accomplished on a per-cell basis as well as being averaged for the entire image (within the masked area). I’m uncertain as to whether a background subtraction step would be necessary.
Ultimately, I would like to output the data to a spreadsheet where I can perform any remaining calculations that are necessary (such as the ratios) and transfer the data into Graphpad/Prism for statistical analysis. Ideally, I would also like to keep saved images of how the cells were masked so that I can attempt to match the setup for future related experiments.
I believe this should process should be fairly reasonable to accomplish, but I am having a great deal of trouble figuring out how to automate the process. Once again, I would greatly appreciate any help and advice you can provide in regard to the best approach to handling this analysis.
Here are some examples of the matching images from each channel. I was only able to upload the individual images as there was an error with the larger stitched images: