Simple quantitation and spatial characterization of label free cellular images.

Label-free imaging is routinely used during cell culture because of its minimal interference with intracellular biology and capability of observing cells over time. However, label-free image analysis is challenging due to the low contrast between foreground signals and background. So far various deep learning tools have been developed for label-free image analysis and their performance depends on the quality of training data. In this study, we developed a simple computational pipeline that requires no training data and is suited to run on images generated using high-content microscopy equipment. By combining classical image processing functions, Voronoi segmentation, Gaussian mixture modeling and automatic parameter optimization, our pipeline can be used for cell number quantification and spatial distribution characterization based on a single label-free image. We demonstrated the applicability of our pipeline in four morphologically distinct cell types with various cell densities. Our pipeline is implemented in R and does not require excessive computational power, providing novel opportunities for automated label-free image analysis for large-scale or repeated cell culture experiments.