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

描述(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它们在许多问题上的适用性。我们假设，一种基于rfCRS的新的分子拉曼显微镜技术将能够检测到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CARS、SRS和SERS，是通过直接探测目标分子的特征来提供分子对比度的方案的子集，避免了外部引入的造影剂引起的潜在并发症。当应用于显微成像时，当前的相干拉曼技术由于其检测低分子浓度的灵敏度而受到限制。我们提出了一种创新的相干拉曼分子成像方法，通过测量向下到射频(RF)光谱范围的微小频率漂移，有望通过拉曼相互作用实现分子检测的数量级更高的灵敏度。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，特别是折射率的时间演化，将光谱移位施加在探测脉冲上。在强、超短激光泵浦脉冲激发分子振动后，将在泵浦-探测配置中测量探测脉冲光谱的变化。我们已经建立了探测脉冲中心频率被相干振动连续调谐--甚至小到频移--而不是典型的散射到由振动拉曼频率分隔的新频率分量的限制。检测非常微小的频移使rfCRS具有极高的灵敏度，因此它能够检测散射组织中极低的分子浓度，接近单分子灵敏度。拟议的项目将审查新的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；以及检测下限和渗透深度的研究。RfCRS所需的关键构建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的应用将极大地扩大可应用相干拉曼显微镜的生物系统的数量？未来的工作将应用体内显微镜研究生物过程，探测和了解疾病过程，评估治疗效果，监测药物植入物释放，以及许多其他方面。由于rfCRS利用了内源振动频谱特征，因此无需开发特定的探头即可应用。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，使其能够检测极低浓度的特定目标分子。我们提出的创新的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可广泛应用于疾病研究、药物治疗疗效的确定和临床诊断程序的具体影响。

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.