# Engineering fluid dynamics of cryo-plunging for improved vitrification

> **NIH NIH R21** · HARVARD UNIVERSITY · 2022 · $225,544

## Abstract

PROJECT SUMMARY ABSTRACT
The long-term goal of this project is to improve cryo-vitrification sample preparation methods for
cryo-electron microscopy (cryo-EM) and tomography (cryo-ET) in terms of their reproducibility
and sample thickness limitations. Cryo-EM is a promising method for observing sub-cellular
assemblies in situ with molecular resolution. However, cryo-EM is hampered by the
irreproducibility and sample thickness limitations imposed by the cryo-vitrification process.
Currently, vitrification is typically achieved by plunging the sample into a cryogenic fluid. This
process of cryo-plunging remains notoriously irreproducible even in structural biology
applications: many cryo-plunging attempts are typically required to get high-quality amorphous
ice. In cell biology applications, the problem is exacerbated: the low thermal diffusivity of cells
puts stringent requirements on the cooling rate in the vitrification process, limiting the thickness
of the sample to the micron scale (<~10 μm), which restricts the application of this technique to
sparsely seeded cells. The cryo-vitrification process will continue to limit the scope and throughput
of cryo-EM until we rigorously understand the fluid dynamics of the sample-cryogen interaction
during cryo-plunging. Once this process is understood, we can engineer it to achieve fast and
reproducible cooling of thicker samples. Optimizing the cryo-vitrification process will address
several critical technical barriers, including: (i) enabling high-throughput sample processing by
increasing the reproducibility of sample preparation, (ii) expanding the scope of cryo-ET by
increasing the thickness of samples eligible for cryo-plunging, and even (iii) achieving time-
resolved nanoscale imaging of biological processes by cooling samples at precise time intervals
after stimulation. The PIs form a collaborative team that is uniquely positioned to address these
technical barriers by using a combination of computational and experimental methods to
understand cryogenic flow and extend the capabilities of cryo-plunging by (1) developing
computational tools to simulate cryo-plunging, (2) systematically exploring the design space and
making testable predictions of system performance, (3) developing and validating a time-resolved
temperature monitoring system, and using it to (4) test theoretical predictions using biological
samples. Upon completion, we will have performed theory-driven experiments evaluating the
most promising cryo-plunging protocols for biological samples. The new protocols will increase
the reproducibility of cryo-plunging and extend this technique to thicker samples, which is
desirable for investigation of biologically relevant cellular assemblies and cell-cell communication.

## Key facts

- **NIH application ID:** 10430822
- **Project number:** 1R21GM146127-01
- **Recipient organization:** HARVARD UNIVERSITY
- **Principal Investigator:** Maxim Prigozhin
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $225,544
- **Award type:** 1
- **Project period:** 2022-09-21 → 2024-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10430822

## Citation

> US National Institutes of Health, RePORTER application 10430822, Engineering fluid dynamics of cryo-plunging for improved vitrification (1R21GM146127-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10430822. Licensed CC0.

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