Discrimination between single Escherichia coli cells using time-resolved confocal spectroscopy.
We describe a technique for rapidly discriminating between single-cell populations within a flowing microfluidic stream. Single-cell time-correlated single-photon counting (scTCSPC) as well as photon burst spectroscopy are used to characterize individual Escherichia coli cells expressed with either green, cyano, or yellow fluorescent protein. The approach utilizes standard confocal fluorescence microscopy incorporating femtoliter detection volumes. The measured burst width characteristics are predominately governed by the fluorescence quantum yield and absorption cross section of the proteins used. It is these characteristics which were used to distinguish between cells with high precision. By utilizing scTCSPC individual fluorescence lifetimes originating from single cells could also be determined. Average fluorescence lifetimes are determined using standard deconvolution procedures. The simplicity of the approach for obtaining well-defined burst width distributions is expected to be extremely valuable for single-cell sorting experiments.
Edel
Joshua B
JB
Institute of Biomedical Engineering, Department of Chemistry, South Kensington, London, SW7 2AZ, United Kingdom.
Lahoud
Pedro
P
Cass
Anthony E G
AE
deMello
Andrew J
AJ
D004926:Escherichia coli / Q000737:chemistry / Q000166:cytology*; D008164:Luminescent Proteins / Q000737:chemistry*; D018613:Microscopy, Confocal / Q000295:instrumentation / Q000379:methods; D012680:Sensitivity and Specificity; D013997:Time Factors
D008164:Luminescent Proteins