Method and apparatus for imaging of cells for counting cells, confluence measurement and plaque detection

An apparatus for measuring cells and cell confluence comprising;

• an imager for imaging a z-stack of bright field images and at least one processor for selecting a best focus bright field image at a z position of f microns in the z-stack of images;
• wherein the at least one processor selects a bright image n microns lower in the z-stack than the best focus bright field image wherein a dot with high contract appears in the center of cells and a dark image m microns higher in the stack than the best focus bright field image wherein the cells as a whole are darker than the background and have a high contrast boundary;
• wherein the at least one processor applies edge detection to the dark image to obtain an edge image and subtracts the edge image from the dark image;
• wherein the at least one processor applies an adaptive threshold to the bright image to create a mask based upon the dots in the cells and uses the adaptive thresholded image as seeds to evaluate the edge image with a watershed analysis to find cell candidates.

A method for measuring cell culture confluence comprising the steps of:

• obtaining an out of focus image above a best focus image of the cell culture wherein a bright spot appears at the center of cells in the image;
• obtaining an out of focus image below the best focus image of the cell culture wherein a reduced brightness appears in areas over the cells in the image;
• applying a threshold to the image above the best focus image to turn the bright spots white;
• using the bright spots of the image above the best focus as seeds for a watershed analysis of the image below the best focus image to create a mask region for each of the areas of reduced brightness and wherein the areas between the mask regions are black;
• and
• identifying all non-cell areas and calculating the area thereof, wherein the confluence is the total area of the cell culture less the area of the non-cell areas.