13 July 2007 Approaches to quantitative single-molecule studies in living cells
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The approach of modeling intracellular networks of biochemical reactions in systems biology demands novel methods suited for acquiring quantitative data about transport and interaction of proteins and metabolites within the heterogeneous environment of living cells. Single-molecule fluorescence spectroscopy (SMFS) has proven a valuable tool for investigating complex structures and processes in biochemistry and molecular biology providing a rich set of methods for in vitro studies of protein/protein and protein/DNA interactions. Although especially designed to reveal spatial and temporal heterogeneities, few applications of SMFS to living cells were reported. Multi-parameter approaches like spectrally-resolved fluorescence lifetime imaging microscopy (SFLIM) have the unique advantage of acquiring individual photons from single molecules carrying along characteristic properties of the fluorescence emission, such as fluorescence lifetime and fluorescence emission spectrum. Herein we present different possibilities for data analysis that can be applied to single molecule data recorded on single photon bases. The photons be used to generate microscopic images with additional contrast based on different fluorescence lifetimes or emission spectra. Information about dynamic processes influencing the characteristic fluorescence signal, like local quenching or molecular diffusion, can be investigated by use of auto- or cross-correlation or on the basis of individual events. Additional information about the number of molecules contributing to the fluorescence emission can be obtained by use of photo-antibunching. Yet the versatile methods of SMFS need further development in view of acceleration and automation of data acquisition and analysis to meet the demands of their application in systems biology.
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Christian Roth, Christian Roth, Pia Heinlein, Pia Heinlein, Dirk-Peter Herten, Dirk-Peter Herten, } "Approaches to quantitative single-molecule studies in living cells", Proc. SPIE 6633, Biophotonics 2007: Optics in Life Science, 663304 (13 July 2007); doi: 10.1117/12.727974; https://doi.org/10.1117/12.727974

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