Abstract
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.
Highlights
The localization and activity of intracellular molecules have been successfully monitored with chimeric fluorescence proteins at single-cell levels to reveal how they dictate cellular function and fate [1,2,3]
Intensity-based software packages have been developed with enriched functionalities with graphic user interfaces [7,8,9,10], while some general open-source ratiometric image analysis tools can be used for time-course quantifications with programming and customization [11, 12]
The Fluocell image analysis software package is designed for the ratiometric quantification of livecell imaging data such as those recorded with two different fluorescent protein-tagged molecules or a Förster or fluorescence (FRET)-based biosensor
Summary
The localization and activity of intracellular molecules have been successfully monitored with chimeric fluorescence proteins at single-cell levels to reveal how they dictate cellular function and fate [1,2,3]. Existing ratiometric analysis tools lack desired flexibility in preprocessing and quantification options and have not been widely used [13, 14] At this front, we developed the Fluocell software package to visualize and quantify dynamic sequences of ratiometric image data with subcellular resolutions and in high throughput. The donor/acceptor emission ratio of the biosensor signals represent local biosensor phosphorylation mediated by the specific kinase in live cells These biosensors can be genetically engineered to localize at different subcellular compartments, including the plasma membrane micro-domains, and to provide versatile measurement of local molecular activities [6, 28]. We describe the systematic design, functional characterization, and application with specific biological problems
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