# Development of Laser-Based Phase Contrast for Biological Electron Microscopy

> **NIH NIH R01** · UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB · 2020 · $437,710

## Abstract

PROJECT SUMMARY
Recent technological advances in transmission electron microscopy of frozen-hydrated specimens (cryo-EM)
have made it possible to retrieve the three-dimensional structure of biological macromolecules with near-
atomic resolution. In conventional cryo-EM, the thin, transparent protein assemblies are made visible by
defocusing the imaging system. The drawback of this method is that the low spatial frequency components of
the image, which are essential for identification and classification of the particles, remain heavily attenuated.
Poor contrast at low spatial frequencies makes it difficult to reconstruct protein complexes with a molecular
weight below 100-200 kDa, or even larger assemblies that exhibit significant structural variability. On the other
hand, cryo-EM reconstruction of particles as small as 40 kDa is theoretically possible with an in-focus phase
contrast device, such as a Zernike phase plate. Recently, the capabilities of Zernike phase contrast in cryo-EM
single particle analysis have been demonstrated with a “Volta” phase plate, based on a thin amorphous carbon
foil. However, a potential for improvement still exists, in particular in achieving a constant, stable phase shift.
In this project, we set out to build a Zernike phase plate for transmission electron microscopy (TEM) based on
a concept borrowed from the field of atomic physics: coherently controlling the motion of quantum particles with
lasers. In this approach, a laser beam focused in the back focal plane of a TEM objective lens creates an
effective potential, which retards the phase of the transmitted wave relative to the scattered wave and thus acts
as a Zernike phase plate. Since no material objects are inserted in the beam path, a laser-based electron
phase plate is not susceptible to electron beam damage. Importantly, it allows for a stable, controllable phase
The necessary high-intensity, continuous laser focus will be created
by amplifying a laser beam in a near-concentric Fabry–Pérot optical resonator with a small mode waist, which
we have developed in our recent work. While we have already demonstrated a sustained optical intensity
sufficient to retard a 300 keV electron beam by 9°, a full 90° phase shift, optimal for a Zernike phase plate, is
well within reach with state of the art cavity mirrors. Our numerical simulations show that with a cavity-based
laser phase plate, phase contrast extends to sufficiently low spatial frequencies to allow for nearly full-contrast
imaging of protein complexes smaller than 5-10 nm. The initial development and characterization of the laser
phase plate's efficacy as a tool for protein reconstruction will take place in a custom-built TEM, designed
specifically for phase plate development. We will then proceed to build a laser phase plate compatible with
standard cryo-EM equipment, with the goal of making the laser-based Zernike phase contrast technology
shift to be applied to the transmitted wave.
available to the br...

## Key facts

- **NIH application ID:** 9951063
- **Project number:** 5R01GM126011-04
- **Recipient organization:** UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
- **Principal Investigator:** Holger Mueller
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $437,710
- **Award type:** 5
- **Project period:** 2017-09-15 → 2021-09-19

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9951063

## Citation

> US National Institutes of Health, RePORTER application 9951063, Development of Laser-Based Phase Contrast for Biological Electron Microscopy (5R01GM126011-04). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9951063. Licensed CC0.

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