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High-throughput single-cell fluorescence spectroscopy.

Dragan Isailovic·Hung-Wing Li·Gregory J Phillips·Edward S Yeung
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Abstract

A high-throughput method for measuring single-cell fluorescence spectra is presented. Upon excitation with a 488 nm argon-ion laser many bacterial cells were imaged by a 20x microscope objective while they moved through a capillary tube. Fluorescence was dispersed by a transmission diffraction grating, and an intensified charge-coupled device (ICCD) camera simultaneously recorded the zero and the first orders of the fluorescence from each cell. Single-cell fluorescence spectra were reconstructed from the distance between zero-order and first-order maxima as well as the length and the pixel intensity distribution of the first-order images. By using this approach, the emission spectrum of E. coli cells expressing green fluorescent protein (GFP) was reconstructed. Also, fluorescence spectra of E. coli cells expressing non-fluorescent apo-subunits of R-phycoerythrin (R-PE) were recorded after incubation of the cells with phycoerythrobilin (PEB) chromophore. The fluorescence spectra are in good agreement with results obtained on the same cells using a fluorescence spectrometer or a fluorescence microscope. When spectra are to be acquired, this approach has a higher throughput, better sensitivity, and better spectral resolution compared to flow cytometry.

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Bacterial ProteinsCell Culture TechniquesEquipment DesignEquipment Failure AnalysisEscherichia coliFlow CytometryGreen Fluorescent ProteinsMicroscopy, FluorescencePhycobilinsPhycoerythrinPyrrolesRecombinant ProteinsReproducibility of ResultsSensitivity and SpecificitySpectrometry, FluorescenceTetrapyrrolesEvaluation StudyJournal ArticleResearch Support, U.S. Gov't, Non-P.H.S.Validation Study

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Dragan Isailovic, Hung-Wing Li, Gregory J Phillips, Edward S Yeung. 2005. High-throughput single-cell fluorescence spectroscopy.. https://doi.org/10.1366/0003702053085124

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