SBIR Phase I: In situ vitrification to unlock routine atomic-scale imaging of biomolecule structure and chemistry in three dimensions

This Small Business Innovation Research Phase I project investigates feasibility of a new microscope sample vessel technology to provide the world’s first routine capability for in situ vitrification and imaging (vitrification on the microscope stage). Ex situ vitrification, such as plunge freezing, is a well-known bottleneck to the speed and quality of vitrified sample studies in cryo-electron microscopy (cryo-EM), cryo-X-ray tomography, and atom probe tomography (APT). Compounding the problem, ex situ vitrification equipment is inaccessible to most researchers due to high costs ranging from tens of thousands to millions of dollars. The new sample vessel will be formed in a confined and ready-to-image geometry, avoiding air-water interface damage and eliminating the need for blotting, milling, and cutting. Successful development will make ex situ vitrification obsolete for many applications, enabling a 100-fold reduction in costs and a 20-fold improvement in time-to-data. Combined with APT, the new sample vessel will provide the first routine capability for imaging the structure and chemistry of biomolecules in 3D. A breakthrough that can be used streamline computer-aided drug design (CADD) by providing data that can reduce pharmaceutical development costs by up to half, a savings of up to $1 billion per drug. The intellectual merit of this project resides in the innovative methods and transformative potential of proving a new microscope sample vessel technology that can physically and chemically suppress ice nucleation in order to provide routine in situ vitrification and imaging. Ex situ vitrification, such as plunge freezing, has had major scientific impact, as recognized with the 2017 Nobel Prize. Successful in situ vitrification development will make ex situ vitrification obsolete for many applications. In previous work, combining the new sample vessel with APT permitted collecting world-first three-dimensional atom maps of mononucleosomes. Phosphorous atoms in the backbone of DNA were used to measure the double-helix structure with an average resolution of 3.8 Ångstroms. Broader application of new sample vessel faces two challenges: (1) low filling success, for e.g., 20 days to image mononucleosomes, and (2) an inability to achieve 1.5 Ångstrom resolution, a key target for CADD. This project aims to: (1) achieve 1 day time-to-data by employing front filling, graphene sealing, and functionalizing a superhydrophilic lumen; and (2) attain 1.5 Ångstrom resolution using a laser matching scission energy of the dominant O-H bond. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.