Enabling Robust Single-Molecule Vibrational Spectroscopy Through Microcavity-Enhancement Single-molecule measurements provide a powerful means for learning about biochemical transformations by allowing multi-step unsynchronized dynamics to be directly observed. However, most existing methods for sin- gle-molecule observations rely on fluorescence. While such experiments excel at determining distances between parts of a biomolecule or binding interactions, they lack the chemical specificity to elucidate, for example, acti- vation or distortion of a particular chemical bond. Such information, which would revolutionize how biological mechanisms are determined and interpreted, could be provided by vibrational spectroscopy. Vibrational spec- troscopy could also eliminate the need for bulky fluorescent dye molecules. However, while methods exist for doing single-molecule vibrational spectroscopy, mainly surface-enhanced Raman spectroscopy (SERS), they are not suited for probe of biologically relevant molecules, as the method requires intimate contact between the part of the biomolecule to be probed (which may be at the interior), and a metal surface or nanoparticle. That metal nanoparticle requires precise alignment relative to the biomolecule and typically causes substantial deformations of the biomolecule of interest, making SERS a substantially invasive and perturbative measurement for biomol- ecules. Thus, a new method is needed to access this highly desirable biochemical information. We propose the development of single-molecule optical microcavity-enhanced Raman spectroscopy. The method will take advantage of the Purcell Effect, which can significantly enhance the Raman scattering cross- section. This effect is maximized in optical microcavities with small mode volumes and high Quality factors, whose use the PI has pioneered for single-particle and single-molecule electronic spectroscopy. Most im- portantly, while SERS requires essentially Van der Waals contact with a plasmonic particle or surface, the optical microcavity enhancement can act at a distance of up to ~100 nm from a dielectric surface, making it suitable for probing biomolecules without significant perturbation, even in the molecular interior. In Specific Aim 1, we will demonstrate single-molecule Raman spectroscopy on an ultrahigh-Q toroidal microcavity, which offers record high Quality factors and small mode volumes. In Specific Aim 2, we will demonstrate single-molecule Raman spectroscopy on a nanobeam resonator, which offers high Quality factors and record small mode volumes. We calculate that microcavity enhancement can lead to a 106 fold increase in signal over state-of-the-art Raman scat- tering spectroscopy. The proof of concept experiments described in this proposal will evaluate the prospects of microcavity-en- hancement as a means to access robust single-molecule vibrational spectroscopy. The long-term objective of this proposal is to bring a new, highly informative, and even disruptive biophysical technique to bear on biological molecules to understand how they operate, change in time, are regulated, and fail.