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Monitoring intracellular oxidative events using dynamic spectral unmixing microscopy
Citation:
Samet, J., W. Cheng, AND J. Larson. Monitoring intracellular oxidative events using dynamic spectral unmixing microscopy. METHODS. Elsevier B.V., Amsterdam, Netherlands, 66(2):345-52, (2014).
Impact/Purpose:
Work describes new method for spectral analysis of cellular processes in living cells
Description:
There is increasing interest in using live-cell imaging to monitor not just individual intracellular endpoints, but to investigate the interplay between multiple molecular events as they unfold in real time within the cell. A major impediment to simultaneous acquisition of multiple fluorescent signals is that many fluorophores emit spectra that overlap, often with maxima that are only a few nanometers apart. Spectral acquisition of mixed fluorescence signals captured within a dedicated scanning range can be used to quantitatively separate signals into component spectra. We report here the novel development of a live cell application of spectral unmixing for the simultaneous monitoring of intracellular events reported by closely emitting fluorophores responding dynamically to external stimuli. We test the performance dynamic spectral unmixing microscopy (DynSUM) using genetically encoded sensors to characterize redox changes and H2O2 production in living cells exposed to control agents as well as an environmental oxidant.