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Related Experiment Video

Updated: Nov 19, 2025

Rejection of Fluorescence Background in Resonance and Spontaneous Raman Microspectroscopy

Rejection of Fluorescence Background in Resonance and Spontaneous Raman Microspectroscopy

Published on: May 18, 2011

Defocus-dependent Thon-ring fading.

Robert M Glaeser¹, Wim J H Hagen², Bong-Gyoon Han¹

¹Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720, USA.

Ultramicroscopy

|February 1, 2021

Summary

This summary is machine-generated.

Defocus-dependent Thon-ring fading in electron cryo-microscopy is caused by Fourier component delocalization. Correcting for magnification anisotropy allows higher defocus values for improved particle imaging and Ewald sphere correction.

Keywords:

Delocalization High defocus Spatial coherence

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Area of Science:

Structural Biology
Microscopy Techniques

Background:

Modern Schottky field-emission guns provide high spatial coherence for electron cryo-microscopy.
Defocus-dependent Thon-ring fading is a common observation, leading to restricted data collection strategies.

Purpose of the Study:

To reproduce and quantitatively analyze defocus-dependent Thon-ring fading.
To explain the causes of Thon-ring fading in single-particle electron cryo-microscopy.
To provide guidelines for optimizing defocus values in imaging.

Main Methods:

Reproduced Thon-ring fading under typical single-particle electron cryo-microscopy conditions.
Performed quantitative analysis of Thon-ring fading.
Applied corrections for linear magnification anisotropy.

Main Results:

Identified delocalization of high-resolution Fourier components as a major cause of Thon-ring fading.
Demonstrated the necessity of correcting for linear magnification anisotropy.
Found that source coherence is a negligible cause of fading under typical conditions.

Conclusions:

Higher defocus values can be utilized in electron cryo-microscopy than previously thought.
Delocalization of Fourier components ultimately limits achievable defocus.
Increased understanding enables confident use of higher defocus for better particle visibility and Ewald sphere correction.