Three-dimensional cell counting for in-situ microscopy

In this contribution, an algorithm is presented for counting cells in the first-, second- and third-layers of three-dimensional three-layer cell clusters from a static intensity image captured by an in-situ microscope. The number of cells in an arbitrary cell cluster is estimated as the ratio between the sum of the areas of the image regions of the parallel projections of the first-, second- and third-layers of the cluster into the image plane and the image area of a circle with a radius equal to the average cell radius of the cluster. The experimental results revealed that counting the cells also in the second- and third-layers of the clusters improved the cell count in 56% in high cell concentrations.