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Nat Protoc. 2016 Apr;11(4):664-87. doi: 10.1038/nprot.2016.036. Epub 2016 Mar 10.

Using Raman spectroscopy to characterize biological materials.

Author information

Lancaster Environment Centre, Lancaster University, Lancaster, UK.
Centre for Global Eco-Innovation, Lancaster Environment Centre, Lancaster University, Lancaster, UK.
Department of Chemistry, Lancaster University, Lancaster, UK.
Daylight Solutions, San Diego, California, USA.
Diamond Light Source, Harwell Science and Innovation Campus, Chilton, Oxfordshire, UK.
Department of Biomedical Physics, Physics and Astronomy, University of Exeter, Exeter, UK.
Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan, USA.
Department of Biomedical and Life Sciences, School of Health and Medicine, Lancaster University, Lancaster, UK.
School of Pharmacy and Biomedical Sciences, University of Central Lancashire, Preston, UK.
Department of Pathology, University of Illinois at Chicago, Chicago, Illinois, USA.
Department of Bioengineering, University of Illinois at Chicago, Chicago, Illinois, USA.
Biophotonics Research Unit, Gloucestershire Hospitals NHS Foundation Trust, Gloucester, UK.


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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