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Updated: Sep 10, 2025
05:33
Light-Induced In Situ Transmission Electron Microscopy for Observation of the Liquid-Soft Matter Interaction
Published on: July 26, 2022
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Liquid Phase Biological Electron Microscopy: Many Published Results and Claimed Benefits Are Fantasy, Not Fact
Robert M Glaeser1, Ravi R Sonani2, Edward H Egelman2
1Department of Molecular and Cell Biology, University of California, 16 Barker Hall #3206, Berkeley, CA 94720, USA.
Summary
Electron microscopy in liquid phase faces resolution limits due to radiation damage and Brownian motion. Analysis shows previously claimed liquid-phase images of GroEL were actually air-dried and negatively stained.
Area of Science:
- Structural biology
- Biophysics
- Electron microscopy
Background:
- Imaging biological molecules in liquid phase via electron microscopy (EM) is desirable for observing dynamic processes.
- High-resolution imaging is challenged by radiation inactivation of molecular function and Brownian motion of suspended particles.
- Previous studies claimed nanometer resolution for liquid-phase EM of macromolecular complexes, but these claims are re-evaluated here.
Purpose of the Study:
- To critically assess the feasibility of high-resolution electron microscopy of biological molecules in their native liquid phase.
- To investigate the cause of contrast reversal in previously published liquid-phase EM images of GroEL.
- To determine if claimed liquid-phase EM images accurately represent molecules in a hydrated state.
Main Methods:
- Analysis of publicly available electron microscopy images of GroEL.
- Comparison of image characteristics with expected outcomes for liquid-phase versus air-dried and stained samples.
- Evaluation of contrast mechanisms in electron microscopy, considering electron scattering by water, ice, and salts.
Main Results:
- Publicly available EM images of GroEL, purportedly from liquid phase, show clear signs of air drying.
- The observed contrast reversal is not due to water scattering but is consistent with negative staining, likely from buffer salts.
- Brownian motion and radiation damage fundamentally limit achievable resolution in true liquid-phase EM.
Conclusions:
- Achieving high-resolution EM imaging of biological macromolecules in a freely suspended liquid phase is currently precluded by physical limitations.
- Re-analysis of key published data suggests that previously reported liquid-phase EM images were artifacts of sample preparation, specifically air drying and negative staining.
- Further methodological advancements are required to overcome radiation damage and motion blur for true in-situ liquid-phase EM at high resolution.
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