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Related Concept Videos

Super-resolution Fluorescence Microscopy01:37

Super-resolution Fluorescence Microscopy

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Super-resolution fluorescence microscopy (SRFM) provides a better resolution than conventional fluorescence microscopy by reducing the point spread function (PSF). PSF is the light intensity distribution from a point that causes it to appear blurred. Due to PSF, each fluorescing point appears bigger than its actual size, and it is the PSF interference of nearby fluorophores that causes the blurred image. Various approaches to achieving higher resolution through SRFM have recently been...
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Cryo-electron Microscopy01:28

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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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Electron Microscope Tomography and Single-particle Reconstruction01:07

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Transmission electron microscopy (TEM) can be used to determine the 3D structure of biological samples with the help of techniques such as electron microscope tomography and single-particle reconstruction. While single-particle reconstruction can examine macromolecules and macromolecular complexes in vitro conditions only, tomography permits the study of cell components or small cells in vivo.
Electron Tomography
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Confocal Fluorescence Microscopy01:16

Confocal Fluorescence Microscopy

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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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Updated: Jun 27, 2025

Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina
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Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina

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Toward quantitative super-resolution methods for cryo-CLEM.

Laura C Zanetti-Domingues1, Michael Hirsch1, Lin Wang1

  • 1CLF Octopus Facility, UKRI-Science and Technology Facilities Council, R92, Rutherford Appleton Laboratory, Didcot, United Kingdom.

Methods in Cell Biology
|May 5, 2024
PubMed
Summary
This summary is machine-generated.

Super-resolution cryogenic fluorescence microscopy advances structural biology by enabling precise 3D localization of low-abundance targets. This technique enhances cryo-electron tomography by integrating functional data with ultrastructural information for comprehensive biological insights.

Keywords:
3D STORMBacterial biologyCLEMClustering analysisDrift correctionMembrane proteinsSTORMSuperSIL STORMcryo-CLEMcryo-STORMcryo-fluorescence microscopy

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Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions
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Sample Preparation by 3D-Correlative Focused Ion Beam Milling for High-Resolution Cryo-Electron Tomography
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Area of Science:

  • Structural Biology
  • Microscopy Techniques
  • Biophysics

Background:

  • Cryo-electron tomography (cryo-ET) revolutionized biological structure determination but is limited to abundant or unambiguous structures.
  • Accurate 3D localization and functional annotation of low-abundance species are crucial for a complete understanding of structure-function relationships.
  • Current fluorescence imaging at cryogenic conditions lacks the resolution for precise localization and functional assignment.

Purpose of the Study:

  • To present the development of super-resolution cryogenic fluorescence techniques for enhanced biological imaging.
  • To demonstrate the application of these techniques for studying biological systems at the ultrastructural level.
  • To discuss the potential of these methods for future cryo-correlative light and electron microscopy (cryo-CLEM) workflows.

Main Methods:

  • Development and application of super-resolution cryogenic fluorescence techniques: superSIL-STORM and astigmatism-based 3D STORM.
  • Imaging of biological systems, including mammalian cells and Escherichia coli.
  • Correlation of functional annotations with ultrastructural information.

Main Results:

  • Demonstration of super-resolution cryogenic fluorescence microscopy for precise 3D localization of biological targets.
  • Application to various biological systems, highlighting the capability to study low-abundance species.
  • Discussion of the advantages and limitations of the presented super-resolution techniques.

Conclusions:

  • Super-resolution cryogenic fluorescence microscopy is essential for realizing the full potential of cryo-ET.
  • These techniques enable the integration of functional and ultrastructural data for comprehensive biological insights.
  • Future applications in cryo-CLEM workflows promise significant advancements in studying complex biological processes, such as membrane protein complexes.