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

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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.