11 February 2011 Simultaneous measurements of fluorescence lifetimes, anisotropy, and FRAP recovery curves
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Abstract
We present fluorescence lifetime imaging (FLIM) and fluorescence anisotropy imaging along with translational diffusion measurements of living cells labelled with green fluorescent protein (GFP) recorded in a single experiment. The experimental set-up allows for time and polarization-resolved fluorescence images to be measured in every frame of a fluorescence recovery after photobleaching (FRAP) series. We have validated the method using rhodamine 123 in homogeneous solution prior to measurements of living A431 cells labelled with cdc42-GFP, for which the FRAP recovery exhibits an immobile fraction and the rotational mobility of the protein is hindered while the fluorescence lifetime fairly homogeneous across the cell. By eliminating the need for sequential measurements to extract fluorescence lifetimes and molecular diffusion coefficients we remove artefacts arising from changes in sample morphology and excessive photobleaching during sequential experiments.
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James A. Levitt, Pei-Hua Chung, Dominic R. Alibhai, Klaus Suhling, "Simultaneous measurements of fluorescence lifetimes, anisotropy, and FRAP recovery curves", Proc. SPIE 7902, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues IX, 79020Y (11 February 2011); doi: 10.1117/12.875151; https://doi.org/10.1117/12.875151
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