Biophotonics: Frequency-modulated Raman Spectroscopy of Biological Specimens

生物光子学：生物样本的调频拉曼光谱

• 批准号: 0086797
• 负责人: Andrew Berger
• 金额: $ 22.23万
• 依托单位: University of Rochester
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2005-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0086797&HistoricalAwards=false
• 关键词: Biophotonics; Frequency; modulated; Raman; Spectroscopy

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.

0086797 BergerVibrational拉曼光谱是一种用于分子种类鉴定和定量的精确工具。由于谱带较窄，可以同时分辨和分析多个组分的信号。 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组分浓度的方法。 这些特性在医学领域的体内和体外测量中都是有价值的。拉曼光谱的获取受到固有样品自荧光效应的限制。 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），并且荧光散粒噪声可以是噪声的限制源。 因此，荧光限制了拉曼光谱作为一种更广泛应用于生物医学目的的技术。研究人员提出开发一种新的基于波长移动的拉曼光谱系统，以抑制这些荧光效应。 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。 该技术使用的激光器，其波长在kHz频率下在1 nm的范围内变化。由于拉曼光谱随着激光波长移动而荧光保持稳定，因此可以有效地拒绝荧光背景;同时，相对于DC检测，kHz范围内的检测显著降低了荧光散粒噪声。 到目前为止，已公布的波长移位拉曼光谱的实现使用稳态检测，其中噪声抑制的优势是丢失的，或单波长扫描，这使得它们太慢，无法与目前的多像素系统竞争。 拟议的系统将结合联合收割机的所有优势，并应显着改善浓度预测。 此外，荧光信号和噪声的抑制可以为生物医学拉曼光谱学开辟新的途径，如可见光激发实验（由于极高的荧光而避免）和更高对比度的拉曼成像。该提议被分解为四个具体目的，它们是（1）组装上述拉曼系统并获得第一轮光谱;（2）优化系统的信噪比和背景信噪比，并与其它拉曼模式进行比较，（3）通过空间傅里叶变换技术提高光谱仪的光通量;以及（4）进行实验以检测生物样品和体模中的临床相关分析物。该提议将基本光谱学与先进仪器相结合，为培养生物医学光学专业的研究生和本科生提供了一个坚实的平台。 通过拉曼光谱学，将出现吸收，荧光和扩散光子迁移的相关主题的讨论。 研究生的会议出席和演讲（明确要求提供资金）将进一步增加对该领域的接触。 在实验室内，学生将获得直接的经验组装激光器，光学，光谱仪和探测器到一个功能系统，并编程计算机来控制系统。 这将培养广泛适用的技能，供未来在学术和工业实验室环境中使用。