Figure 2. Cell localization, contour detection, and separation of aggregated cells.

(A) Based on negative phase contrast image, peaks of light intensity are detected for all cells, as indicated by red circles. (B) Preliminary masks are obtained with the threshold method. Masks in green are areas with multiple peaks indicating aggregated cells. Masks in yellow are areas with single cell. (C) The boundaries of the preliminary masks are extracted to serve as initial contours for individual cells. (D) The contour expansion method is applied to detect cell boundaries for all cells in the field of view. (E) Steps taken to separate three aggregated cells selected in figure (A). The first figure shows three peaks detected, indicating three cells in the selected area. The second figure shows the mask area defined by the threshold method and locations of three detected peaks. The third figure shows division of the mask area into three sub-areas based on shortest distance between any given pixels and the three detected peaks. The fourth figure shows contour initiation by extracting the outlines of sub-areas. The fifth figure shows the final contours of three cells after contour expansion.