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    The phase separation and aggregation of proteins are hallmarks of many neurodegenerative diseases. These processes can be studied in living cells using fluorescent protein constructs and quantitative live-cell imaging techniques, such as fluorescence recovery after photobleaching (FRAP) or the related fluorescence loss in photobleaching (FLIP). While the acquisition of FLIP images is straightforward on most commercial confocal microscope systems, the analysis and computational modeling of such data is challenging. Here, a novel model-free method is presented, which resolves complex spatiotemporal fluorescence-loss kinetics based on dynamic-mode decomposition (DMD) of FLIP live-cell image sequences. It is shown that the DMD of synthetic and experimental FLIP image series (DMD-FLIP) allows for the unequivocal discrimination of subcellular compartments, such as nuclei, cytoplasm, and protein condensates based on their differing transport and therefore fluorescence loss kinetics. By decomposing fluorescence-loss kinetics into distinct dynamic modes, DMD-FLIP will enable researchers to study protein dynamics at each time scale individually. Furthermore, it is shown that DMD-FLIP is very efficient in denoising confocal time series data. Thus, DMD-FLIP is an easy-to-use method for the model-free detection of barriers to protein diffusion, of phase-separated protein assemblies, and of insoluble protein aggregates. It should, therefore, find wide application in the analysis of protein transport and aggregation, in particular in relation to neurodegenerative diseases and the formation of protein condensates in living cells.


    Daniel Wüstner. Dynamic Mode Decomposition of Fluorescence Loss in Photobleaching Microscopy Data for Model-Free Analysis of Protein Transport and Aggregation in Living Cells. Sensors (Basel, Switzerland). 2022 Jun 23;22(13)

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    PMID: 35808232

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