Biophotonics: Frequency-modulated Raman Spectroscopy of Biological Specimens

University Of Rochester, Rochester NY

Investigators

Abstract

0086797 Berger Vibrational Raman spectroscopy is a precise tool for the identification and quantification of molecular species. Because the spectral bands are narrow, signals from many compo-nents can be resolved and analyzed simultaneously. Consequently, Raman spectroscopy provides a method for analyzing multiple components' concentrations in complex sam-ples accurately, non-invasively, and non-destructively. These attributes are valuable in the medical field for both in vivo and in vitro measurements. Acquisition of Raman spectra is limited by the effects of intrinsic sample autofluor-escence. For most biological samples, the spectral background from fluorescence will typically be much (greater than100 times) larger than the Raman signal, and fluorescence shot noise can be the limiting source of noise. Consequently, fluorescence limits Raman spec-troscopy from being a more widely applicable technique for biomedical purposes. The investigators propose to develop a novel Raman spectroscopy system based upon wavelength shifting to suppress these fluorescence effects. Such a system would be able to acquire fluorescence-corrected Raman spectra with unprecedented speed and accuracy. The technique uses a laser whose wavelength is varied over a range of < 1 nm at kHz fre-quencies. Because Raman spectra shift with the laser wavelength while fluorescence re-mains stable, the fluorescence background can be efficiently rejected; at the same time, detection in the kHz regime reduces the fluoresence shot noise significantly relative to DC detection. To date, the published implementations of wavelength-shifted Raman spectroscopy have used either steady-state detection, in which the noise-suppression advantage is lost, or single-wavelength scanning, which makes them too slow to com-pete with present multipixel systems. The proposed system would combine all of the ad-vantages and should significantly improve concentration predictions. In addition, sup-pression of fluorescence signal and noise could open new avenues for biomedical Ra-man spectroscopy, such as visible-excitation experiments (avoided because of extremely high fluorescence) and higher-contrast Raman imaging. This proposal is broken into four Specific Aims, which are (1) to assemble the above Raman system and acquire a first round of spectra; (2) to optimize the signal-to-back-ground and signal-to-noise of the system and compare values to other Raman modali-ties; (3) to increase the optical throughput of the spectrometer via spatial Fourier-trans-form techniques; and (4) to conduct experiments to detect clinically relevant analytes in biological samples and phantoms. This proposal combines basic spectroscopy with advanced instrumentation, provid-ing a solid platform for training graduate and undergraduate students in biomedical optics. Through Raman spectroscopy, discussion of related topics in absorption, fluo-rescence, and diffuse photon migration will arise. Conference attendance and presen-tations by graduate students (for which funding is explicitly requested) will further in-crease exposure to the field. Within the laboratory, students will gain direct experience assembling lasers, optics, spectrographs, and detectors into a functioning system and also programming computers to control the system. This will nurture broadly applica-ble skills for future use in both academic and industrial laboratory settings.