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Raman tweezers sorting of single microbial cells
Author(s) -
Huang Wei E.,
Ward Andrew D.,
Whiteley Andrew S.
Publication year - 2009
Publication title -
environmental microbiology reports
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.229
H-Index - 69
ISSN - 1758-2229
DOI - 10.1111/j.1758-2229.2008.00002.x
Subject(s) - pseudomonas fluorescens , raman spectroscopy , yeast , microorganism , single cell analysis , microbiology and biotechnology , spheroplast , chemistry , optical tweezers , escherichia coli , biology , biophysics , bacteria , cell , biochemistry , optics , genetics , physics , gene
Summary We have selectively isolated microbial cells by identifying and then manipulating cells using a combination of Raman microspectroscopy and optical trapping. The criterion for cell discrimination is based on spectral peak shifts within the Raman spectrum of individual cells. A specific shift in the phenylalanine peak position from 1001 rel. cm −1 to 965 rel. cm −1 is utilized to indicate the uptake of 13 C within the cell that utilized 13 C‐substrate. Cells were captured and manipulated using an infrared (1064 nm) laser while Raman spectra were acquired over shorter timescales (30 s) using a co‐aligned 514.5 nm laser beam. Selected cells were manoeuvred to a clean part of a capillary tube and the tubes were cleaved to physically separate the cells. The technique was tested for cell viability and cross‐contamination effects using 70 single yeast cells ( Saccharomyces cerevisia ). Following these tests, 58 single bacterial cells ( Escherichia coli DH5α, and Pseudomonas fluorescens SBW25::Km‐RFP) that exhibited 13 C uptake were sorted from bacterial populations. Among those isolated cells, 11 out of 18 yeast cells and 7 out of 18 single SBW25::Km‐RFP cells were recovered by incubation; 2 out of 7 sorted yeast cells and 3 out of 8 sorted bacterial cells (single SBW25::Km‐RFP) were genome amplified correctly. We show that the Raman tweezers approach has the potential to open a new frontier to study unculturable microorganisms, which account for more than 99% microbes in natural environment.