# Visualizing Live Cell Physiology with High Resolution Using Phase-Contrast STEM

> **NIH NIH R01** · UNIVERSITY OF NOTRE DAME · 2022 · $479,590

## Abstract

Project Summary
 This proposal describes a plan to develop a method for visualizing live cell physiology with high resolution
using integrated Differential Phase Contrast-Scanning Transmission Electron Microscopy (iDPC-STEM) at low
dose to promote viability. Visualizing physiology demands spatial resolution with a commensurate depth-of-field on the scale of the protein machinery (3-7 nm) that drives it without concomitant damage. With the
introduction of a liquid flow cell containing water in a vacuum-tight envelope made from membranes that are
transparent to the electron beam, it should be possible to scrutinize biology with high-resolution under
physiological conditions with STEM. This proposal focuses on three specific technical challenges, testing
solutions in a crucible of well characterized biological systems:
1. Improve resolution using a liquid flow cell formed from ultra-thin membranes and thin spacers. To
reduce scattering in the membrane and liquid, it is practical to shrink the silicon nitride (SiN) membranes
forming the liquid cell to 8-10 nm, and space them 150 nm apart without compromising the window integrity. To
eliminate bulging in a liquid cell loaded with fluid, the windows will be reinforced with thick ribs so that a large
>400 "mu"m2 area can be spanned. However, even 10 nm SiN membranes are still too thick for high-resolution
imaging. So, (3 nm) thin amorphous silicon (a-Si) and atomically thin graphene or h-BN membranes spanning
ribs formed from SiN will be used as windows for high-resolution imaging. The resolution will be tested using a
Titan STEM by visualizing adenosine triphosphate (ATP) and fluorescent streptavidin (STR).
2. Improve contrast with iDPC-STEM imaging. To increase the visibility of transparent biological samples, a
phase-contrast method for imaging, iDPC-STEM, will be adopted that uses a four-quadrant (segmented) split-
detector to measure the gradient of a phase object. iDPC-STEM boasts a higher signal to noise ratio compared
to conventional STEM, which offers the possibility for extremely low-dose imaging. The resolution, contrast and
concomitant damage will be tested in an aberration-corrected, iDPC-equipped Themis Z (with 60 pm
resolution) by visualizing ATP and fluorescent STR in thin (0-50 nm thick) liquid layers.
3. Finally, low-dose iDPC-STEM will be used with an ultra-thin liquid flow cell to visualize the smallest
prokaryotic cells. If the electron probe interacts with a cell at the top membrane in the liquid cell, high-resolution images may be captured this way. Because the probe is so shallow along the optic-axis, a focus
series may also be used to section a cell for 3D tomography. To test these ideas, four strains of Mycoplasma
(100 nm in size) will be cultured in a shallow (150 nm) flow cell and visualized with iDPC-STEM to discover the
role their nanostructure plays in infection. In specimens this thick, multi-slice simulations may be required to
inform on the structure. After exposure to ...

## Key facts

- **NIH application ID:** 10485182
- **Project number:** 5R01GM138870-03
- **Recipient organization:** UNIVERSITY OF NOTRE DAME
- **Principal Investigator:** GREGORY LOUIS TIMP
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $479,590
- **Award type:** 5
- **Project period:** 2020-08-01 → 2024-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10485182

## Citation

> US National Institutes of Health, RePORTER application 10485182, Visualizing Live Cell Physiology with High Resolution Using Phase-Contrast STEM (5R01GM138870-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10485182. Licensed CC0.

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