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Raman tweezers sorting of single microbial cells

Authors

  • Wei E. Huang,

    Corresponding author
    1. Molecular Microbial Ecology, CEH-Oxford, Mansfield Road, Oxford OX1 3SR, UK.
      *E-mail w.huang@sheffield.ac.uk; Tel. (+44) 114 2225796; Fax (+44) 114 2225701. † Present address: Kroto Research Institute, University of Sheffield, Broad Lane, Sheffield S3 7HQ, UK.
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  • Andrew D. Ward,

    1. Science and Technology Facilities Council, Rutherford Appleton Laboratory, Harwell Science and Innovation Campus, Didcot OX11 0QX, UK.
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  • Andrew S. Whiteley

    1. Molecular Microbial Ecology, CEH-Oxford, Mansfield Road, Oxford OX1 3SR, UK.
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*E-mail w.huang@sheffield.ac.uk; Tel. (+44) 114 2225796; Fax (+44) 114 2225701.

Present address: Kroto Research Institute, University of Sheffield, Broad Lane, Sheffield S3 7HQ, UK.

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.

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