Systems and methods for counting cells

A system, comprising:

• an imaging system configured to produce an image of a plurality of cells in a vessel in a plurality of focal planes;
• at least one controller coupled to the imaging system;
• and
• the at least one controller coupled to memory containing instructions that when executed:
• control the imaging system to capture a plurality of z-stack images of at least some cells, each of the plurality of z-stack images including two-dimensional x-y images, at different focal lengths, corresponding to focal planes at different z coordinates relative to the imaging system, to capture focused images of each of the at least some cells;
• in each of the z-stack images, determine an area of at least one cell by separating a foreground of the image from a background of the image and producing a binary mask that indicates for each pixel whether it is in the foreground or background, wherein the pixels in the foreground correspond to a focused image of a cell,
• identify objects in the foreground that have a size of at least one cell and identify objects having a size greater than that of one cell as a cell cluster;
• for each object determined to be a cell cluster, find at least one sharp peak in an intensity contour in the object which corresponds to a center of a cell;
• classify objects in the focused image as live cells or dead cells;
• and
• count the number of cells by counting the number of objects identified as live single cells in all of the plurality of images, counting the number of sharp peaks in the objects identified as a live cell cluster in all of the plurality of images, and counting the number of objects classified as dead cells.