Biophotonics: Frequency-modulated Raman Spectroscopy of Biological Specimens

振动拉曼光谱是鉴定和定量分子种类的精确工具。由于光谱带较窄，因此可以同时解析和分析来自多个组分的信号。因此，拉曼光谱提供了一种准确、非侵入性和非破坏性地分析复杂样品中多种成分浓度的方法。这些属性对于体内和体外测量在医学领域都是有价值的。拉曼光谱的获取受到样品自身荧光效应的限制。对于大多数生物样品，来自荧光的光谱背景通常比拉曼信号大得多（大于100倍），荧光散点噪声可能是噪声的限制源。因此，荧光限制了拉曼光谱技术在生物医学领域的广泛应用。研究人员建议开发一种基于波长移动的新型拉曼光谱系统来抑制这些荧光效应。这样的系统将能够以前所未有的速度和精度获得荧光校正的拉曼光谱。该技术使用的激光波长在千赫频率下在1nm范围内变化。由于拉曼光谱随激光波长移动而荧光保持稳定，因此可以有效地抑制荧光背景；与此同时，相对于直流检测，在kHz波段检测可显著降低荧光散粒噪声。迄今为止，已发表的波长移拉曼光谱的实现要么使用稳态检测，在这种检测中失去了噪声抑制优势，要么使用单波长扫描，这使得它们太慢，无法与现有的多像素系统竞争。拟议的系统将结合所有的优点，并应显著改善浓度预测。此外，抑制荧光信号和噪声可以为生物医学拉曼光谱开辟新的途径，例如可见激发实验（由于极高的荧光而避免）和高对比度拉曼成像。该方案分为四个具体目标，即：(1)组装上述拉曼系统并获得第一轮光谱；(2)对系统的信本和信噪进行优化，并与其他拉曼模态进行比较；(3)利用空间傅里叶变换技术提高光谱仪的光通量；(4)进行实验，检测生物样品和幻影中的临床相关分析物。本方案将基础光谱学与先进仪器相结合，为培养生物医学光学研究生和本科生提供了坚实的平台。通过拉曼光谱，将讨论吸收、荧光和漫射光子迁移等相关主题。研究生的会议出席和演讲（明确要求资金）将进一步增加对该领域的接触。在实验室中，学生将获得将激光、光学、光谱仪和探测器组装成一个功能系统以及编写计算机程序来控制系统的直接经验。这将培养广泛适用的技能，为未来在学术和工业实验室环境中使用。

0086797BergerVibrational 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.