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| - Discrimination between Single Escherichia coli Cells Using Time-Resolved ConfocalSpectroscopy
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| Abstract
| - We describe a technique for rapidly discriminating between single-cell populations within a flowing microfluidicstream. Single-cell time-correlated single-photon counting (scTCSPC) as well as photon burst spectroscopyare used to characterize individual Escherichia coli cells expressed with either green, cyano, or yellowfluorescent protein. The approach utilizes standard confocal fluorescence microscopy incorporating femtoliterdetection volumes. The measured burst width characteristics are predominately governed by the fluorescencequantum yield and absorption cross section of the proteins used. It is these characteristics which were usedto distinguish between cells with high precision. By utilizing scTCSPC individual fluorescence lifetimesoriginating from single cells could also be determined. Average fluorescence lifetimes are determined usingstandard deconvolution procedures. The simplicity of the approach for obtaining well-defined burst widthdistributions is expected to be extremely valuable for single-cell sorting experiments.
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