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它们在许多问题上的适用性。我们假设基于rfCRS的一种新的分子拉曼显微镜技术将能够在散射组织中检测极低的分子浓度（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特征来提供分子对比的方案的一个子集，避免了外部引入的造影剂引起的潜在并发症。当应用于显微镜成像时，当前的相干拉曼技术受到其检测低分子浓度的灵敏度的限制。我们提出了一种相干拉曼分子成像的创新方法，该方法有望通过测量拉曼相互作用（低至射频（rf）光谱范围及以下）的小频移，实现更高数量级的分子检测灵敏度。因此我们称之为射频相干喇曼光谱（rfCRS）。光谱位移是通过目标分子的光学性质，特别是折射率的时间演变施加在探测脉冲上的。在强、超短的激光泵浦脉冲激发分子振动后，将在泵浦-探针结构中测量探针脉冲光谱的变化。我们已经确定，探针脉冲中心频率可以通过相干振动连续调谐，甚至可以降低到很小的频率位移，而不受典型的散射限制，由振动拉曼频率分离到新的频率分量。检测非常小的频移使rfCRS具有极高的灵敏度，从而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；并对其检出限和渗透深度进行了研究。rfCRS所需的关键构建模块已经在我们的实验室进行了测试。如果这个假设被证明是正确的，rfCRS的应用将极大地扩展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来研究生物过程，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利用了内源振动谱特征，因此无需开发专用探针即可应用。因此，对健康科学的影响可能是深远的。

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.