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Using Raman spectroscopy to characterize biological materials

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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Figure 1: Schematic identifying light scattering after laser exposure on a sample surface.
Figure 2: Raman microspectroscopy workflow diagram highlighting the key aspects of experimental design including instrumentation, sample preparation, spectral acquisition and data processing with representative examples.
Figure 3: Generalized overview of instrumentation options within a typical spontaneous Raman spectroscopic microscope system.
Figure 4: Simplified overview of the effect of laser excitation wavelength on the fluorescence background.
Figure 5: Raman spectra derived from blood serum containing 150- and 40-nm diameter gold nanospheres, and a control sample containing no SERS substrates.
Figure 6: Examples of common troubleshooting issues during spectral acquisition.
Figure 7: A brief overview of pre-processing options in Raman spectral data analysis and their contribution to spectral transformation using an example spectrum from a tomato plant leaflet.
Figure 8: Raman map of fixed endometrial tissue, focusing on uterine glands that spiral throughout the tissue.

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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).

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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.

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

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Butler, H., Ashton, L., Bird, B. et al. Using Raman spectroscopy to characterize biological materials. Nat Protoc 11, 664–687 (2016). https://doi.org/10.1038/nprot.2016.036

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