Hi I am new to using cell analysis software. I am trying to find the percentage of cells in a DAPI image that are arrested in mitotic phase. I primarily need the software to count the number of cells in the image. The images are all in blue. I have attempted to do a pipeline to count the cells. I have also used the practice pipeline. I do not want to count nuclei or secondary objects, merely count the cells. When I try to run a pipeline, it tells me that my image is in color and must be in grayscale. Does the image need to be converted to grayscale? Also an advice on how to create a proper pipeline would be much appreciated as I am quite confused at this time. Thanks so Much!!!
The best help that we can give you is with an example image or two and your current pipeline. Would you please upload these here?
The blue that you see is just a default rendering of your image software. If the pixels are only in the blue channel, then you really just have grayscale images.
Yes, many CellProfiler modules require a grayscale image. Use the ColorToGray module to convert these to grayscale (in your case, use the Split option, and only check the Blue channel and redefine it as, say, “Grayscale”).
The nuclei count is generally the same as the count of the cells, so I am unclear what you mean. Of course, in your case, cells arrested in the mitotic phase might complicate the counting using image analysis. One can often spuriously segment 2 “nuclei”, though they are really one object for each mitotic spindle, depending on the exact phase they are arrested in. Is this what you mean? In that case, it is often helpful to have another, whole cell marker (e.g. phalloidin) to account for the 2N doubling effect. But I really cannot say with any confidence without looking at your images.