# Development of an Instant Freezing Sample Preparation System for Cryo-EM

> **NIH NIH R43** · HUMMINGBIRD PRECISION MACHINE COMPANY · 2022 · $237,305

## Abstract

Project Summary
Project Title: Development of an Instant Freezing Sample Preparation System for Cryo-EM
Company Name: Hummingbird Precision Machine Co., dba Hummingbird Scientific
Principal Investigator: Norman Salmon
Summary:
The field of cryogenic electron microscopy (cryo-EM) has rapidly been gaining traction as a
structural characterization technique for proteins. Sample preparation for cryo-EM is
advantageous over other proven techniques like x-ray crystallography as proteins do not need to
be crystallized for high-resolution structural analysis. While the cryo-EM sample preparation
technique keeps proteins in their native form, cryo-EM suffers from a bottleneck in both quality
and throughput of samples. Recent advances in the field have focused on increasing sample
throughput through automated preparation techniques. However, the more important problem in
cryo-EM samples now has become protein damage from the air-water interface. Before vitreous
freezing, proteins in the sample droplet collide and stick to the droplet surface, where they
denature to form an unraveled monolayer. The more time the sample is exposed before freezing,
the more time proteins have time to interact with the surface and denature. Current commercial
systems fail to directly address the problems associated with the interface, focusing instead on
the automated production of samples with thin, vitreous ice. While these improvements are
valuable to the overall goals of reducing ice thickness, they ignore damage to the sample
molecules themselves, preventing users from repeatably obtaining high-resolution structures.
The proposed project aims to revolutionize sample preparation techniques for cryo-EM by
exploring a method of generating higher quality samples that exhibit significantly less damage
from the air-water interface. The proposed system aims to limit sample exposure to the air-water
interface by reducing the rate of freezing by an order of magnitude. In order to reduce damage
from the air-water interface with high quality samples, the system will aim to vitrify samples in less
than 10 ms with a large percentage of ice suitable for high-resolution imaging. This “fast freezing”
system also is also conceived to populate sample grids with consistently thin ice by reducing the
amount of excess water that is frozen on the grid, creating larger areas of uniform ice and
biomolecular sample per grid. Hummingbird Scientific aims to develop and test the theory behind
the system in Phase I.

## Key facts

- **NIH application ID:** 10384540
- **Project number:** 1R43GM145044-01
- **Recipient organization:** HUMMINGBIRD PRECISION MACHINE COMPANY
- **Principal Investigator:** Norman James Salmon
- **Activity code:** R43 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $237,305
- **Award type:** 1
- **Project period:** 2022-08-01 → 2025-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10384540

## Citation

> US National Institutes of Health, RePORTER application 10384540, Development of an Instant Freezing Sample Preparation System for Cryo-EM (1R43GM145044-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10384540. Licensed CC0.

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