Abstract

Raman spectroscopy can be used to measure the chemical composition of a sample, which can in turn be used to extract biological information. Many materials have characteristic Raman spectra, which means that Raman spectroscopy has proven to be an effective analytical approach in geology, semiconductor, materials and polymer science fields. The application of Raman spectroscopy and microscopy within biology is rapidly increasing because it can provide chemical and compositional information, but it does not typically suffer from interference from water molecules. Analysis does not conventionally require extensive sample preparation; biochemical and structural information can usually be obtained without labeling. In this protocol, we aim to standardize and bring together multiple experimental approaches from key leaders in the field for obtaining Raman spectra using a microspectrometer. As examples of the range of biological samples that can be analyzed, we provide instructions for acquiring Raman spectra, maps and images for fresh plant tissue, formalin-fixed and fresh frozen mammalian tissue, fixed cells and biofluids. We explore a robust approach for sample preparation, instrumentation, acquisition parameters and data processing. By using this approach, we expect that a typical Raman experiment can be performed by a nonspecialist user to generate high-quality data for biological materials analysis.

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Acknowledgements

H.J.B.'s project is mediated by the Centre for Global Eco-Innovation funding through the European Regional Development Fund (ERDF). Work in F.L.M.'s laboratories has been funded by the UK Engineering and Physical Sciences Research Council (EPSRC), the Rosemere Cancer Foundation and the UK Biotechnology and Biological Sciences Research Council (BBSRC). K.E.-W. acknowledges a Pilot and Feasibility grant from the Michigan Diabetes Research and Training (subsidiary of National Institutes of Health (NIH)/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) 2P30 DK020572).

Author information

Affiliations

  1. Lancaster Environment Centre, Lancaster University, Lancaster, UK.

    • Holly J Butler
    • , Pierre L Martin-Hirsch
    • , Martin R McAinsh
    •  & Francis L Martin
  2. Centre for Global Eco-Innovation, Lancaster Environment Centre, Lancaster University, Lancaster, UK.

    • Holly J Butler
  3. Department of Chemistry, Lancaster University, Lancaster, UK.

    • Lorna Ashton
  4. Daylight Solutions, San Diego, California, USA.

    • Benjamin Bird
  5. Diamond Light Source, Harwell Science and Innovation Campus, Chilton, Oxfordshire, UK.

    • Gianfelice Cinque
  6. Department of Biomedical Physics, Physics and Astronomy, University of Exeter, Exeter, UK.

    • Kelly Curtis
    • , Jennifer Dorney
    •  & Benjamin Gardner
  7. Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan, USA.

    • Karen Esmonde-White
  8. Department of Biomedical and Life Sciences, School of Health and Medicine, Lancaster University, Lancaster, UK.

    • Nigel J Fullwood
  9. School of Pharmacy and Biomedical Sciences, University of Central Lancashire, Preston, UK.

    • Pierre L Martin-Hirsch
  10. Department of Pathology, University of Illinois at Chicago, Chicago, Illinois, USA.

    • Michael J Walsh
  11. Department of Bioengineering, University of Illinois at Chicago, Chicago, Illinois, USA.

    • Michael J Walsh
  12. Biophotonics Research Unit, Gloucestershire Hospitals NHS Foundation Trust, Gloucester, UK.

    • Nicholas Stone

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Contributions

F.L.M. is the principal investigator who conceived the idea for and finalized the manuscript; H.J.B. wrote and compiled the manuscript and figures. L.A., B.B., G.C., K.E.-W., B.G., M.J.W., M.R.M. and N.S. provided information and feedback throughout the article; and K.C., J.D., N.J.F. and P.L.M.-H. provided feedback on the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Martin R McAinsh or Nicholas Stone or Francis L Martin.

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DOI

https://doi.org/10.1038/nprot.2016.036

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