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Spatiotemporal regulation of molecular activities dictates cellular function and fate. Investigation of dynamic molecular activities in live cells often requires the visualization and quantitation of fluorescent ratio image sequences with subcellular resolution and in high throughput. Hence, there is a great need for convenient software tools specifically designed with these capabilities. Here we describe a well-characterized open-source software package, Fluocell , customized to visualize pixelwise ratiometric images and calculate ratio time courses with subcellular resolution and in high throughput. Fluocell also provides group statistics and kinetic analysis functions for the quantified time courses, as well as 3D structure and function visualization for ratio images. The application of Fluocell is demonstrated by the ratiometric analysis of intensity images for several single-chain Förster (or fluorescence) resonance energy transfer (FRET)-based biosensors, allowing efficient quantification of dynamic molecular activities in a heterogeneous population of single live cells. Our analysis revealed distinct activation kinetics of Fyn kinase in the cytosolic and membrane compartments, and visualized a 4D spatiotemporal distribution of epigenetic signals in mitotic cells. Therefore, Fluocell provides an integrated environment for ratiometric live-cell image visualization and analysis, which generates high-quality single-cell dynamic data and allows the quantitative machine-learning of biophysical and biochemical computational models for molecular regulations in cells and tissues.

Fluocell for Ratiometric and High-Throughput Live-Cell Image Visualization and Quantitation