I am currently creating a macro which will count the number of foci in each channel of a GFP/RFP fluorescence image of yeast cells after dead cells have been removed from the image. In previous versions of my macro I was using a simple ‘Threshold’ function to find foci, followed by ‘Analyse particles’ to find how many there were. This was proving to be unreliable so I moved onto using the MosaicSuite’s Spot detection plugin (http://mosaic.mpi-cbg.de/?q=downloads/imageJ) to find foci in the image instead. The problem with this plugin seems to be that if I apply a mask to remove dead cells by using the ImageCalculator to multiply the mask by the image, leaving a black area on the image, like so:
The plugin gets confused by the black area and detects many false ‘foci’ around its edge:
According to the documentation for the plugin, there should be the option to add in a mask layer, however I have found no way to apply the mask as there isn’t an apply button!
I imagine there must be a way to get the plugin to only look at a certain area of the image using the ROI manager perhaps, but I have not used this enough to know how to segment an image using it.
I have binary mask files and images to work with. Could someone suggest a way to segment my images using these masks so that the foci counting plugin will work properly?
Thanks for any help,