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 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.
Isailovic
Dragan
D
Ames Laboratory-USDOE and Department of Chemistry, Iowa State University, Ames, Iowa 50011, USA.
Li
Hung-Wing
HW
Phillips
Gregory J
GJ
Yeung
Edward S
ES
D001426:Bacterial Proteins / Q000032:analysis / Q000378:metabolism*; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods; D004867:Equipment Design; D019544:Equipment Failure Analysis; D004926:Escherichia coli / Q000166:cytology / Q000378:metabolism*; D005434:Flow Cytometry / Q000295:instrumentation* / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D052980:Phycobilins; D010799:Phycoerythrin / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D011758:Pyrroles / Q000032:analysis / Q000378:metabolism*; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000295:instrumentation* / Q000379:methods*; D045725:Tetrapyrroles
D001426:Bacterial Proteins; D052980:Phycobilins; D011758:Pyrroles; D011994:Recombinant Proteins; D045725:Tetrapyrroles; D010799:Phycoerythrin; D049452:Green Fluorescent Proteins; C011070:phycoerythrobilin