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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.
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