# Improving cryoEM and cryoFIB performance through amelioration of mechanical stress in vitreous ice.

> **NIH NIH R21** · RUTGERS, THE STATE UNIV OF N.J. · 2021 · $235,500

## Abstract

Project Summary/Abstract
 Stress introduced during vitrification has been proposed as a likely cause of two
experimentally-observed methodological challenges: loss of resolution in cryo-electron
microscopy (cryoEM) and attrition of lamellae milled in cryo-focused ion beam
micromachining (cryoFIB). CryoEM is limited by a loss of information when the first
few electrons strike, and mounting evidence points to beam-catalyzed relaxation of the
built-up stress as the cause. A limitation of cryoFIB is that milled specimens break or
snap during milling because of stress, but the source of this stress has not been shown.
 When specimens are prepared by plunge-freezing, water expands while the
specimen support contracts. Additives that disrupt the open hydrogen-bonding network
of water change its thermal expansivity. We will use volume-modulating excipients to
change the thermal expansion coefficient of cryoEM and cryoFIB samples and eliminate
mechanical stress from this source.
 Rheological properties of ice suggest that thermal annealing of cryoEM grids will
allow solvent to flow and alleviate stress. We will warm cryoEM grids without
devitrifying them to relieve stress.
 Therefore, our goal is to test whether volume-modulating excipients or
thermal annealing will solve the problem of beam-induced motion and
improve performance by 1. increasing resolution in cryoEM and 2. increasing
yield in cryoFIB.

## Key facts

- **NIH application ID:** 10303673
- **Project number:** 1R21GM140345-01A1
- **Recipient organization:** RUTGERS, THE STATE UNIV OF N.J.
- **Principal Investigator:** Jason T Kaelber
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $235,500
- **Award type:** 1
- **Project period:** 2021-09-01 → 2023-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10303673

## Citation

> US National Institutes of Health, RePORTER application 10303673, Improving cryoEM and cryoFIB performance through amelioration of mechanical stress in vitreous ice. (1R21GM140345-01A1). Retrieved via AI Analytics 2026-06-24 from https://api.ai-analytics.org/grant/nih/10303673. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*