High-Throughput Single-Cell Fluorescence Spectroscopy

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 20× microscope objective while they moved through a capillary tube. Fluorescence was dispersed by a transmission diffraction grati...

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Veröffentlicht in:Applied spectroscopy 2005-02, Vol.59 (2), p.221-226
Hauptverfasser: Isailovic, Dragan, Li, Hung-Wing, Phillips, Gregory J., Yeung, Edward S.
Format: Artikel
Sprache:eng
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Zusammenfassung: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 20× 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.
ISSN:0003-7028
1943-3530
DOI:10.1366/0003702053085124