Optical microscopy and spectroscopy are widely used in multiple research areas relating to biology. Label-free spectroscopy and imaging are valuable tools that permit interrogation of biological samples without the need for exogenous labels, allowing for investigation of unperturbed biological systems. We demonstrate a coherent Raman technique called Doppler Raman (DR) spectroscopy which combines impulsive excitation with a novel frequency shift detection scheme for rapid, high sensitivity detection of low to medium frequency vibrational modes from 10-1800cm-1. Briefly, the DR spectroscope is a pump-probe system where the pump beam generates a time-varying index of refraction proportional to the Raman response of the sample. The time-delayed probe beam undergoes a frequency shift in the sample due to the time-varying index of refraction that is resolved using a novel high-sensitivity detection scheme. Other coherent Raman techniques such as Stimulated Raman Scattering (SRS) and Coherent Anti-Stokes Raman Spectroscopy (CARS) have been used to provide sensitive, label-free contrast for an array of biological targets, but their ability to detect low frequency vibrational modes is limited. Biologically significant targets like cytochrome c (740-760cm-1), DNA (782, 788, 1095cm-1), hydroxyapatite, and numerous pharmaceutical drugs exhibit rich Raman spectra across a range of low frequency modes below the well-known “fingerprint region”. Additionally, many proteins like hemoglobin, insulin, and bovine serum albumin have breathing modes below 50cm-1. Sensitive detection of low-frequency Raman vibrational modes unlocks a suite of potential biological and chemical dynamics like protein conformational changes and protein super complex formation.
David R. Smith, David G. Winters, Scott R. Domingue, Jeff Field, Daniel Kane, Jesse W. Wilson, and Randy A. Bartels, "High-sensitivity detection of Raman vibrations in the impulsive limit with Doppler Raman spectroscopy (Conference Presentation)," Proc. SPIE 10890, Label-free Biomedical Imaging and Sensing (LBIS) 2019, 108901M (Presented at SPIE BiOS: February 04, 2019; Published: 4 March 2019); https://doi.org/10.1117/12.2509685.6009876035001.
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