I’m trying to make a pipeline to count cells that are HNA (a nuclear stain for human cells) positive in a graft where cells were injected into rat tissue. When we take the images of these grafts, it is an image made of many tiles stitched together as the total magnification is 200X. So, there are often large areas that are outside of the graft and make the image files unnecessarily large. We had cropped them so that the run time for all the images to be processed by CellProfiler would be faster.
We noticed that the number of objects, however, changes between the original image and the cropped image. For example, for one image, the un-cropped image had about 300 objects identified while the cropped image had only about 200 objects identified. There shouldn’t be that much of a difference as the sections cropped out did not have more than 2 or 3 potential objects in them.
The resolution between cropped and un-cropped images is the same, so it is only the size of the image that is different. The pipeline consists of EnhanceOrSuppressFeatures, ApplyThreshold, and IdentifyPrimaryObjects. Do you know why this might be occurring? Any help would be appreciated! Please let me know if you need any additional information.