High sensitivity, molecular contrast microscopy with radio-frequency Coherent Ram

描述（由申请人提供）：当前相干拉曼光学显微镜技术的分子检测灵敏度限制了其对许多问题的适用性。我们假设，一种新的分子拉曼显微镜技术的基础上，rfCRS将能够检测散射组织中极低的分子浓度（接近单分子灵敏度）。相干拉曼光谱，包括汽车、SRS和Sers，是通过直接探测目标分子的特性来提供分子对比度的方案的子集，避免了由外部引入的对比剂引起的潜在复杂性。当应用于显微成像时，当前相干拉曼技术受到其检测低分子浓度的灵敏度的限制。我们提出了一种创新的相干拉曼分子成像的方法，预计通过测量小的频率偏移-下至射频（rf）光谱范围和以下的拉曼相互作用，实现数量级更高的灵敏度的分子检测。因此，我们称之为射频相干拉曼光谱（rfCRS）.通过目标分子的光学性质（具体地，折射率）的时间演化对探测脉冲施加光谱偏移。在探测脉冲光谱的变化将被测量在一个泵浦探测配置后，分子振动已被激发的强烈，超短激光泵浦脉冲。我们已经确定，探测脉冲中心频率是连续调谐的相干振动-甚至下降到小的频移-没有散射的典型限制，以新的频率成分分离的振动拉曼频率。检测非常小的频移使rfCRS具有极高的灵敏度，因此能够检测散射组织中极低的分子浓度，接近单分子灵敏度。拟议的项目将研究新的rfCRS技术的许多方面：透明样品和散射组织中的分子灵敏度检测;以及检测限和穿透深度的研究。rfCRS所需的关键构建模块已经在我们的实验室进行了测试。如果这一假设被证明是正确的，rfCRS的实用性将极大地扩大相干拉曼显微镜可以应用的生物系统的数量？未来的工作将应用体内显微镜研究生物过程，探测和理解疾病过程，评估治疗效果，监测药物植入物释放等。由于rfCRS利用内源性振动光谱特征，因此无需开发特定探针即可应用。因此，对健康科学的影响可能是有利的。 公共卫生相关性：疾病和药物治疗的分子基础迫使分子成像技术的发展，其具有检测极低浓度的特定靶分子的能力。我们提出的创新rfCRS技术将提高相干拉曼显微镜的检测灵敏度-允许检测前所未有的低浓度分子。rfCRS可广泛应用，对疾病研究、药物治疗疗效测定和临床诊断程序具有特定影响。

DESCRIPTION (provided by applicant): The sensitivity of molecule detection of current coherent Raman optical microscopy techniques limits their applicability to many problems. We hypothesize that a new molecular Raman microscopy technique based on that rfCRS will be able to detect extremely low molecule concentrations (approaching single-molecule sensitivity) in scattering tissue. Coherent Raman optical spectroscopies, including CARS, SRS, and SERS, are a subset of schemes providing molecular contrast by probing the target molecules' characteristics directly, avoiding potential complications arising from externally-introduced contrast agents. When applied to microscopy imaging, current coherent Raman techniques are limited by their sensitivities to detect low molecular concentrations. We propose an innovative approach to coherent Raman molecular imaging that is expected to achieve orders of magnitude higher sensitivity for molecule detection through Raman interactions by measuring small frequency shifts - down to the radio frequency (rf) spectral range and below. Consequently we call this rf coherent Ra- man spectroscopy (rfCRS). The spectral shifts are imposed on a probe pulse by the time evolution of the optical properties of the target molecules, specifically, the refractive index. Changes in the probe pulse spectra will be measured in a pump-probe configuration after molecular vibrations have been excited by an intense, ultra short laser pump pulse. We have established that the probe pulse center frequency is continuously tuned by the coherent vibrations - even down to small frequency shifts - without the typical restriction of scattering to new frequency components separated by the vibration Raman frequency. Detecting very small frequency shifts gives rfCRS its exquisite sensitivity and thus its ability to detect extremely low molecule concentrations, approaching single-molecule sensitivity, in scattering tissue. The proposed project will examine many aspects of the new rfCRS technique: Molecular sensitivity detection in transparent samples and in scattering tissues; and studies of the detection limits and penetration depths. The key building blocks needed for rfCRS have already tested in our laboratory. Should the hypothesis prove true, the utility of rfCRS will tremendously expand the number of biological systems to which coherent Raman microscopy can be applied? Future work will apply in-vivo microscopy for studying biological processes, probing and understanding disease processes, assessing efficacy of treatments, monitoring drug implant release, and many others. Because rfCRS makes use of endogenous vibration spectral features, it can be applied without the need for development of specific probes. As a result, the impact on health sciences could be pro- found. PUBLIC HEALTH RELEVANCE: The molecular foundations of disease and drug treatments compel the development of molecular imaging techniques with the capability to detect extremely low concentrations of specific target molecules. The innovative rfCRS technique that we propose will improve the detection sensitivity of coherent Raman microscopy - allowing detection of unprecedented low concentrations of molecules. rfCRS could be widely applicable with specific impacts on the study of disease, determination of the efficacy of drug treatments, and for clinical diagnostic procedures.