High-throughput microcolony growth analysis from suboptimal low-magnification micrographs

New technological advances have enabled high-throughput phenotyping at the single-cell level, yet analyzing the large amount of data generated by high throughput phenotyping experiments automatically and accurately is a considerable challenge. Here we introduce Processing Images Easily (PIE), software that automatically tracks growth of microbial colonies in low-magnification brightfield images by combining adaptive object-center recognition with gradient-based object-outline recognition. PIE recognizes colony outlines very robustly and accurately across a wide range of image brightnesses, focal depths, and organisms. Beyond accurate colony recognition, PIE is designed to easily integrate with complex experiments, allowing colony tracking across multiple experimental phases and classification based on fluorescence intensity. We show that PIE can be used to accurately measure the growth rates of large numbers (>90,000) of bacterial or yeast microcolonies in a single-time-lapse experiment, allowing calculation of population-wide growth properties. Finally, PIE is able to track individual colonies across multiple experimental phases, measuring both growth and fluorescence properties of the microcolonies. Author Summary High-throughput microscopy has enabled automated collection of large amounts of growth and gene-expression data in microbes. Computational methods that can precisely recognize and track organisms in images are essential to performing measurements at scale using automated microscopy. We have developed PIE, software that automatically recognizes microbial colonies in microscopy images, tracks them in imaging time-series, and performs measurements of growth and, potentially, gene expression. PIE is highly effective on low-resolution images, outperforming current state-of-the-art approaches in both speed and accuracy, and works well in microbes of varying shapes and sizes. In addition, PIE allows tracking microcolonies across arbitrary sequences of experimental phases, each collecting data in different modalities. We show that PIE allows measurement of growth and fluorescence properties in tens of thousands of microbial colonies in a single experiment, and that in turn the scale of these measurements can lead to important insights about interindividual differences in growth and stress response. PIE is available as a Python package () with documentation currently at ; users can also run analysis on individual images or time-series without the need to install PIE by using our web application, currently available at . ### Competing Interest Statement The authors have declared no competing interest.