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In vivo Raman spectroscopy for real-time biochemical assessment of tissue pathology and physiology

Nature Protocols volume 21, pages 1780–1808 (2026)Cite this article

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Abstract

In vivo Raman spectroscopy (RS) enables fast, label-free evaluation of tissue biochemistry in situ with high molecular specificity. The Raman spectrum provides a chemical ‘fingerprint’ of tissue composition, facilitating investigations of dynamic changes in real-time in various physiological and pathophysiological states. This capability makes in vivo RS a promising approach for rapid diagnostics, surgical guidance and biological research. Despite its numerous advantages, the widespread acceptance of RS for in vivo measurements has been hindered by the lack of a standardized stepwise protocol. This protocol serves as a guide for applying RS in vivo and includes steps for proper instrument selection, system alignment, calibration, system parameter setup, in vivo data collection, instrument cleaning, spectral pre-processing, data analysis and interpretation. Troubleshooting information is described for overcoming challenges in acquiring in vivo RS data due to inherently weak Raman signals, variable tissue optical properties, autofluorescence background and interference from ambient lighting and off-target tissues. Specific steps for applying in vivo RS in the skin, cervix, esophagus and colon are described and can be readily adapted to probe other organs. Typical parameters for acquiring and processing in vivo Raman spectra, as well as example spectral output from different organs, are provided for reference. Ultimately, this standardized protocol serves as a guideline to enhance the repeatability of in vivo RS studies and further expand the adoption of this approach as a research and clinical tool.

Key points

  • This protocol details a standardized procedure for conducting in vivo Raman spectroscopy experiments, enabling label-free analysis of tissue biochemistry in situ with high specificity in the presence of water. The protocol includes applications in diagnostics, therapeutic monitoring and biological research in multiple human organs.

  • Alternative label-free methods for acquiring molecular information from biological tissues include autofluorescence spectroscopy, diffuse reflectance spectroscopy and Fourier transform IR spectroscopy.

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Fig. 1: Procedures for performing an in vivo RS study.
Fig. 2: In vivo Raman system instrumentation.
Fig. 3: Troubleshooting for in vivo RS measurements.
Fig. 4: In vivo RS for human skin assessment.
Fig. 5: In vivo Raman spectra of the human cervix.
Fig. 6: In vivo Raman spectra from human gastrointestinal tissue.

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Data availability

Source data for the figures in this protocol are available at figshare.com with the identifier https://doi.org/10.6084/m9.figshare.29474396 (ref. 114). Source data are provided with this paper.

Code availability

Code for Raman data pre-processing is available at figshare.com with the identifier https://doi.org/10.6084/m9.figshare.29474396 (ref. 114) and at https://github.com/MJLabVBC/in-vivo-raman-spectroscopy.

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Acknowledgements

The authors acknowledge J.S. Baba for providing critical feedback on the manuscript.

Author information

Authors and Affiliations

  1. Vanderbilt Biophotonics Center, Vanderbilt University, Nashville, TN, USA

    Ezekiel J. Haugen, Rekha Gautam, Andrea K. Locke & Anita Mahadevan-Jansen

  2. Department of Biomedical Engineering, Vanderbilt University, Nashville, TN, USA

    Ezekiel J. Haugen, Andrea K. Locke & Anita Mahadevan-Jansen

  3. Biophotonics Group, Tyndall National Institute, Cork, Ireland

    Rekha Gautam

  4. Department of Chemistry, Vanderbilt University, Nashville, TN, USA

    Andrea K. Locke

Authors
  1. Ezekiel J. Haugen
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  2. Rekha Gautam
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  3. Andrea K. Locke
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  4. Anita Mahadevan-Jansen
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Contributions

E.J.H., R.G., A.K.L. and A.M.-J. wrote the manuscript. E.J.H. prepared the figures.

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Correspondence to Ezekiel J. Haugen or Anita Mahadevan-Jansen.

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Nature Protocols thanks Surya Pratap Singh and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

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Key references

Haugen, E. J. et al. Sci. Rep. 15, 22471 (2025): https://doi.org/10.1038/s41598-025-05591-z

Rourke-Funderburg, A. et al. Appl. Spectrosc. 79, 1228–1241 (2024): https://doi.org/10.1177/00037028241307043

Locke, A. et al. Clin. Transl. Gastroenterol. 15, e00665 (2024): https://doi.org/10.14309/ctg.0000000000000665

Masson, L. E. et al. Am. J. Obstet. Gynecol. 227, 275.e1–275.e14 (2022): https://doi.org/10.1016/j.ajog.2022.02.019

Pence, I. J. et al. Biomed. Opt. Express 8, 524–535 (2017): https://doi.org/10.1364/BOE.8.000524

Supplementary information

Supplementary Video 1 (download MP4 )

Video demonstrating how to perform alignment of an in vivo Raman system

Supplementary Code 1 (download ZIP )

Scripts for pre-processing in vivo fingerprint and high–wavenumber Raman spectra

Source data

Source Data Fig. 4 (download XLSX )

In vivo fingerprint and high–wavenumber Raman spectra from the skin.

Source Data Fig. 5 (download XLSX )

In vivo fingerprint Raman spectra from the cervix.

Source Data Fig. 6 (download XLSX )

In vivo fingerprint and high–wavenumber Raman spectra from gastrointestinal tissue.

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Haugen, E.J., Gautam, R., Locke, A.K. et al. In vivo Raman spectroscopy for real-time biochemical assessment of tissue pathology and physiology. Nat Protoc 21, 1780–1808 (2026). https://doi.org/10.1038/s41596-025-01274-1

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