Dispersion indices for universal quantification of fluorescently-labelled subcellular structure spatial distributions

Image analysis of subcellular structures and biological processes relies on specific, context-dependent pipelines, which are labor-intensive, constrained by the intricacies of the specific biological system, and inaccessible to broader applications. Here we introduce the application of dispersion indices, a statistical tool traditionally employed by economists, to analyze the spatial distribution and heterogeneity of subcellular structures. This computationally efficient high-throughput approach, termed GRID (Generalized Readout of Image Dispersion), is highly generalizable, compatible with open-source image analysis software, and adaptable to diverse biological scenarios. GRID readily quantifies diverse structures and processes to include autophagic puncta, mitochondrial clustering, and microtubule dynamics. Further, GRID is versatile, applicable to both 2D cell cultures and 3D multicellular organoids, and suitable for high-throughput screening and performance metric measurements, such as half-maximal effective concentration (EC50) values. The approach enables mechanistic analysis of critical subcellular structure processes of relevance for diseases ranging from metabolic and neuronal diseases to cancer as well as a first-pass screening method for identifying biologically active agents for drug discovery.