The primary objects I want to identify are in transiently transfected cells in the green channel. First I wanted to know if anyone has suggestions on how to mask the image (using MaskImage) for different ranges of intensity, and then using those masked images in subsequent modules. I got the maskimage to work, however, when I try and use IdentifyPrimObject only in the image created by the selected objects in a specified range, the identification always gives results in objects outside of the masked image. Currently I am just using crop to rectify this problem, and cropping the image based on my range of expression levels that i want.
Next, I have ran into a wall identifying a specific primary object. Like i said I am identifying primary objects in a channel that is transiently transfected. However, the object that I am identifying is not specifically stained. It is actually the brightest spot within a nucleus (the spot is a multiple copy of promoters). However, the cells that have this bright spot, also have a background intensity in the area of the nucleus that is not the spot (the nucleoplasm), but not always in the same intensity ratio of spot to nucleus. So I used a CorrectIllumination module to take away some of the background, then increased the threshhold on the identifyPrimObjects module, however, we often get false positives and false negatives on the spot identification. Any suggestions would be appreciated. Also if you would like I can send a power point illustrating these questions.