17 February 2009 Application of FRET biosensors and computational analysis for live cell imaging
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Abstract
Biosensors designed on the principle of fluorescent resonance energy transfer (FRET) have been widely applied to visualize signaling cascades in live cells with high spatiotemporal resolution. In this paper, we review the work in our lab related to the application of FRET biosensors in studying molecular events in live cells, and our work using computational analysis methods to explore complex biological information implicated in FRET images. Membrane-tethered Src biosensors were used to visualize the dynamics of Src activity in subcellular microdomains. We have developed a finite element (FE) method to analyze the movement of biosensors. Based on fluorescence recovery after photobleaching (FRAP) experiments, the estimation and subtraction of biosensor diffusion revealed a high Src activity at cell periphery upon growth factor stimulation. In addition to Src, a RhoA biosensor was used to study the subcellular feature of RhoA activity in migrating HeLa cells. We have developed an image registration method to automatically track and quantify the FRET signals within user-defined subcellular regions, and classify the dynamics of subcellular pixels according FRET signals. The results revealed that the RhoA activity is polarized in the migratory cells, with the gradient of polarity oriented toward the opposite direction of cell migration. Therefore, FRET biosensors integrated with computational analysis provide powerful tools to precisely decode the complex dynamics of signaling transduction regulated in subcellular locations of live cells.
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Shaoying Lu, Yingxiao Wang, "Application of FRET biosensors and computational analysis for live cell imaging", Proc. SPIE 7191, Fluorescence In Vivo Imaging Based on Genetically Engineered Probes: From Living Cells to Whole Body Imaging IV, 719108 (17 February 2009); doi: 10.1117/12.812183; https://doi.org/10.1117/12.812183
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