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

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.