Integrated Mixing and Inkjet Deposition for Time Resolved Cryo-EM Sample Preparation

About This Grant

Project Summary Project Title: Integrated Mixing and Inkjet Deposition for Time Resolved cryo-EM Sample Preparation Company Name: Hummingbird Precision Machine Co., dba Hummingbird Scientific Principal Investigator: Norman Salmon Summary: This project aims to develop, for the first time, an integrated mixing, inkjet deposition, and gas- assisted rapid freezing system for time-resolved cryogenic electron microscopy (cryo-EM) sample preparation, enabling the capture of short-lived biomolecular states essential for understanding biomolecular dynamics. Cryo-EM provides atomic-resolution structures of biomolecules in their native hydrated states, but current sample preparation methods take several seconds from sample application to freezing—too slow to capture short-lived intermediates that exist only on the millisecond timescales. Accessing these intermediates is essential for elucidating disease mechanisms, advancing therapeutics, and designing more efficient enzymes. To address this gap, this new system will combine microfluidic mixing with our patent pending methods for precision inkjet dispensing and rapid cold-gas assisted freezing into a compact sample preparation platform compatible with standard cryo-EM workflows. The system features an inkjet printhead with an integrated micromixer for on-demand mixing, followed by programmable picolitre droplet deposition in a defined pattern with millisecond-scale temporal control. The deposited samples are rapidly vitrified using a cold gas flow, eliminating the variability and inconsistencies of conventional blotting and plunge freezing methods that utilize liquid cryogens. This approach enables preparation of cryo-EM grids at precisely defined reaction time points, allowing structural characterization of dynamic molecular transitions and direct visualization of short-lived intermediates that are otherwise inaccessible with current methods. The project’s long-term goal is to transform structural biology by providing researchers with a tool to capture biomolecules in motion on the millisecond timescale. To achieve this, the specific aims include optimizing micromixer design and integration, refining piezoelectric dispensing electronics and control software, integrating sample freezing hardware, and validating system performance through cryo-EM imaging of test samples. By enabling routine capture of short-lived intermediates, this technology will support NIH’s mission to advance biomedical research and therapeutic development, with broad applications in drug discovery, disease mechanism elucidation, and enzyme engineering.