Rapid 3D phenotypic analysis of neurons and organoids using data-driven cell segmentation-free machine learning

Phenotypic profiling of large three-dimensional microscopy data sets has not been widely adopted due to the challenges posed by cell segmentation and feature selection. The computational demands of automated processing further limit analysis of hard-to-segment images such as of neurons and organoids. Here we describe a comprehensive shallow-learning framework for automated quantitative phenotyping of three-dimensional (3D) image data; using unsupervised data-driven voxel-based feature learning, which enables computationally facile classification, clustering and advanced data visualization. We demonstrate the analysis potential on complex 3D images by investigating the phenotypic alterations of: neurons in response to apoptosis-inducing treatments and morphogenesis for oncogene-expressing human mammary gland acinar organoids. Our novel implementation of image analysis algorithms called Phindr3D allowed rapid implementation of data-driven voxel-based feature learning into 3D high content analysis (HCA) operations and constitutes a major practical advance as the computed assignments represent the biology while preserving the heterogeneity of the underlying data. Phindr3D is provided as Matlab code and as a stand-alone program (https://github.com/DWALab/Phindr3D).Author summary: Fluorescence microscopy is a fundamental technology for cell biology. However, unbiased quantitative phenotypic analysis of microscopy images of cells grown in 3D organoids or in dense culture conditions in large enough numbers to reach statistical clarity remains a fundamental challenge. Here, we report that using data driven voxel-based features and machine learning it is possible to analyze complex 3D image data without compressing them to 2D, identifying individual cells or using computationally intensive deep learning techniques. Further, we present methods for analyzing this data by classification or clustering. Together these techniques provide the means for facile discovery and interpretation of meaningful patterns in a high dimensional feature space without complex image processing and prior knowledge or assumptions about the feature space. Our method enables novel opportunities for rapid large-scale multivariate phenotypic microscopy image analysis in 3D using a standard desktop computer.