Cell segmentation image processing methods

A method for segmenting cell colonies in an image of cell colonies, comprising the steps of:

• providing a stack of images of cells;
• calculating a variance image that displays the variance of each pixel position for the whole stack, wherein pixels with the highest variance are the ones having different values across the whole stack;
• thresholding the variance image to create a cell mask of the pixels that are dark at the bottom of the stack, transparent in the middle, and bright at the top of the stack;
• forming an average image of all the images in the stack, wherein each pixel position of the average image holds the average of all the pixels for its corresponding position in the image stack;
• forming a synthetic background image by calculating the median pixel color of all the pixels that are not on the mask and if a pixel in the average image is darker than a darkness threshold value or brighter than a brightness threshold value, it is changed to a median value;
• calculating a synthetic background grayscale histogram of the synthetic background image;
• calculating a pixel grayscale histogram of the pixels under the cell mask;
• and
• determining a best focus when the curve of the pixel grayscale histogram is closest to the curve of the synthetic background grayscale histogram and using the histograms to create an image wherein the cell colonies are segmented.