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Raman tweezers sorting of single microbial cells.

Huang, Wei E.; Ward, Andrew D.; Whiteley, Andrew S.. 2009 Raman tweezers sorting of single microbial cells. Environmental Microbiology Reports, 1 (1). 44-49. 10.1111/j.1758-2229.2008.00002.x

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Abstract/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 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.

Item Type: Publication - Article
Digital Object Identifier (DOI): 10.1111/j.1758-2229.2008.00002.x
Programmes: CEH Topics & Objectives 2009 onwards > Biodiversity > BD Topic 2 - Ecological Processes in the Environment
CEH Sections: Hails
NORA Subject Terms: Biology and Microbiology
Ecology and Environment
Date made live: 12 Nov 2009 12:57
URI: http://nora.nerc.ac.uk/id/eprint/8291

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