With advances in detector technology and software algorithms, single-particle cryo- electron microscopy (cryo-EM) has taken the structural biology field by storm in the past five years. As the demand for three-dimensional reconstruction of biomacromolecules grows exponentially in both the academic and industrial sectors, technology features such as high throughput, high resolution, and automation have become increasingly important. However, significant improvements in the upstream portion of the cryo-EM workflow especially sample preparation on metal gridsare still lacking. The current state-of-the- art solution to this problem is rather unsatisfactory and presents many technical issues ranging from a lack of user control on sample thickness to a long exposure of protein molecules at the hydrophobic water-air interface before vitrification. This small business innovation research project proposes to tackle these technical challenges by making use of the state-of-the-art microfluidic technology in vacuum. This enables the protein samples to be deposited and vitrified on cryo-EM grids 1000x faster than the current standard, thereby requiring only very small amounts of samples in total for grid preparation. Furthermore, the new sample preparation stage offers advanced users the ability to adjust sample thickness based on their experimental needs, as well as perform time-resolved measurements on the proteins and their interactions with one another. To better understand and characterize the technical capabilities and limitations of the proposed innovation for the cryo-EM sample preparation market, additional research and development work will be needed before a beta prototype can be built for in-lab testing and solicitation of user feedback. The approximate timeline for technology validation, iterative design, and commercialization should take no less than three years, with technology validation being the deliverable at the end of Phase I SBIR and product validation and iterative design in Phase II. This project addresses broad problems across the field of structural biology. By providing scientists a reliable, reproducible, and repeatable method to prepare biological samples for cryo-electron microscopy studies, this project enables them to gain a better understanding of the structural heterogeneity and conformational flexibility of proteins and protein complexes in microbial and plant systems in their natural environment. This new knowledge facilitates as well as accelerates the research development in bioenergy, biofuel, and sustainability and thus greatly benefits human lives in the long run.
