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Related Concept Videos

Cryo-electron Microscopy01:28

Cryo-electron Microscopy

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Conventional electron microscopy (EM) involves dehydration, fixation, and staining of biological samples, which distorts the native state of biological molecules and results in several artifacts. Also, the high-energy electron beam damages the sample and makes it difficult to obtain high-resolution images. These issues can be addressed using cryo-EM, which uses frozen samples and gentler electron beams. The technique was developed by Jacques Dubochet, Joachim Frank, and Richard Henderson, for...
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Confocal microscopy is an advanced microscopic technique. The prime advantage of the confocal microscope over other microscopy techniques is its ability to block the out-of-focus light from the illuminated samples using pinholes. It is widely used with fluorescence optics to obtain high-resolution, sharp contrast images. Unlike optical microscopes, confocal microscopes use a focused beam of light laser to scan the entire sample surface at different z-planes. These microscopes are, therefore,...
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Related Experiment Video

Updated: Sep 12, 2025

Sample Preparation by 3D-Correlative Focused Ion Beam Milling for High-Resolution Cryo-Electron Tomography
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Systematic Characterization of Optical Aberrations Reveals Cryo-FLM Localization Fidelity.

Hongjia Li, Lauren Ann Metskas, Fang Huang

    Biorxiv : the Preprint Server for Biology
    |August 6, 2025
    PubMed
    Summary

    Cryo-correlative light and electron microscopy (cryo-CLEM) faces optical aberrations limiting molecular localization accuracy. Mitigating these distortions with point spread function modeling improves precision to under ten nanometers.

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    Area of Science:

    • Cell Biology
    • Structural Biology
    • Microscopy

    Background:

    • Cryo-correlative light and electron microscopy (cryo-CLEM) integrates fluorescence and electron microscopy for in situ cellular analysis.
    • Combining single-molecule localization with cryo-CLEM offers high-resolution molecular positioning within cellular ultrastructure.
    • Cryogenic fluorescence light microscopy (cryo-FLM) is hampered by optical aberrations, affecting localization accuracy and downstream cryo-electron microscopy.

    Purpose of the Study:

    • To systematically analyze optical aberrations in a commercial cryo-FLM system.
    • To identify sources of optical distortions affecting point spread function (PSF) accuracy.
    • To evaluate strategies for mitigating localization errors in cryo-FLM.

    Main Methods:

    • Quantitative analysis of optical aberrations in a commercial cryo-FLM system.
    • Investigation of system imperfections, refractive index mismatches, and sample heterogeneities as aberration sources.
    • Application of spatially matched and adaptive PSF models to correct for optical distortions.

    Main Results:

    • Significant optical aberrations were identified, causing lateral errors up to 90 nm and axial errors over 300 nm.
    • These aberrations stem from system imperfections, refractive index mismatches, and sample heterogeneities.
    • PSF modeling reduced localization errors to ten nanometers or less, significantly improving accuracy.

    Conclusions:

    • Accurate, in situ PSF modeling is crucial for achieving nanometer-scale molecular localization in cryo-FLM.
    • The developed experimental pipeline serves as a tool for assessing optical performance in cryo-CLEM and related workflows.
    • Aberration-aware cryo-FLM and cryo-CLEM strategies are essential for precise molecular positioning in vitrified specimens.