Time-gated confocal Raman microscope

描述(申请人提供)：生命科学的进步取决于新工具和仪器的发展。我们在细胞和分子水平上了解生命系统功能的能力大大增强，因为成像技术能够提供体内的结构和化学信息。拉曼光谱是真正的非侵入性的，它可以提供关于生物组织的化学成分和物理结构的重要信息。这项小规模(R03)研究计划旨在开发一种非侵入性显微光谱学的创新方法，以便明确地探索细胞和组织中化学成分的变化。为了实现这一目标，PI将利用时间门控拉曼成像的概念，首次将其应用于显微镜，以实现对荧光背景具有优越分辨率的共焦成像。预计它将在信噪比方面提供至少一个数量级的改善，这将转化为至少100倍的采集速率，同时提高空间分辨率并消除在解析复杂拉曼波段方面可能存在的模糊性。第一个具体目标是建立一种新型仪器的原型并表征其性能。第二个具体目标是为几个模型系统初步测试一种新的分子、细胞和组织成像仪器：1)胶原自组织和结构变化，导致骨结构发育；2)视网膜色素上皮细胞的光降解，这被认为是导致失明的主要原因；3)牙齿组织中的早期龋病检测。许多医学研究和诊断应用需要侵入性最小的显微分子成像。拉曼显微光谱学是一种非侵入性的、化学特异性的分子成像技术，但由于强烈的荧光背景，导致信噪比显著降低，难以揭示振动带的精细结构。提出的研究为显微成像提供了一个解决方案，通过在特殊设计的光学布置中对有用信号进行时间选通来解决这一问题。

DESCRIPTION (provided by applicant): Progress in the Life Sciences depends upon the development of new tools and instruments. Our ability to understand the function of living systems on a cellular and molecular level is greatly enhanced by imaging techniques capable of providing structural and chemical information in vivo. Raman spectroscopy is truly non-invasive, and it could provide significant information on the chemical composition and physical structure of biological tissues. This small-scale (R03) research proposal aims to develop an innovative approach for non-invasive microspectroscopy in order to unambiguously explore changes in chemical composition in cells and tissues. To achieve this goal, the PI will utilize the concept of time-gated Raman imaging, applied for the first time to microscopy to achieve confocal imaging with superior discrimination against the fluorescence background. It is expected that it will provide at least an order of magnitude improvement in the signal-to-noise ratio, which transforms into at least 100 times faster acquisition rates, while improving spatial resolution and eradicating possible ambiguities in resolving complex Raman bands. The first specific aim is building the prototype of a novel instrument and characterizing its performance. The second specific aim is pilot testing of a new instrument for molecular, cellular and tissue imaging for several model systems: 1) collagen self-organization and structural transformation, which leads to bone structure development, 2) photodegradation of retinal pigment epithelium cells, which is considered to be the major cause of blindness, and 3) early caries detection in dental tissues. Many medical research and diagnostic applications require microscopic molecular imaging with minimum invasiveness. Raman microspectroscopy, being a non-invasive, chemically specific technique for molecular imaging, suffers a dramatic drawback due to a strong fluorescent background, which significantly reduces the signal-to-noise ratio and makes it difficult to reveal the fine structure of vibrational bands. The proposed research provides a solution to this problem for microscopic imaging by time-gating the useful signal in a specially designed optical arrangement.