Transforming single-molecule mass measurement by rotating total internal reflection mass photometry

We aim to develop a novel label-free optical microscopy approach to transform our ability to characterize and quantify biomolecular dynamics and interactions at the single small-molecule level. The core of the technology, termed rotating total internal reflection mass photometry (RoMP), rests on delivering a step change in label-free detection and quantification of light scattering from individual biomolecules in solution. We will achieve this by leveraging the advantages of total internal reflection to boost optical contrast, and suppress background signatures. This will be enabled for the first time in the context of interferometric microscopy through the use of a specifically-designed optical attenuation optic. Compared to the state-of-the-art Mass Photometry (MP), RoMP will transform the quantitative detection limit and improve mass resolution from ~50 kDa to 10 kDa and from 20 kDa to 2 kDa, respectively. These advances will dramatically expand the range and identity of studies that can be performed by single molecule mass measurement, representing an advance comparable to the change in resolution from 15 to 1.5 Angstrom in cryo-electron microscopy over the past decade. The interdisciplinary and complementary aspects foster two-way transfer of optical nano-imaging knowledge between the applicant (illumination-modulated fluorescence microscopy) and the host (detection-modulated label-free mass microscopy), ultimately catalyzing innovative biochemical investigations and their application by a broad user base for maximum impact.