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Raman tweezers sorting of single microbial cells.

By Wei E. Huang, Andrew D. Ward and Andrew S. Whiteley

Abstract

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 13C within the cell that utilized 13C-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 13C 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

Topics: Biology and Microbiology, Ecology and Environment
Year: 2009
DOI identifier: 10.1111/j.1758-2229.2008.00002.x
OAI identifier: oai:nora.nerc.ac.uk:8291
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