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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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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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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.
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A scanning electron microscope (SEM) is used to study the surface features of a sample by using an electron beam that scans the sample surface in a two-dimensional manner. Typically, areas between ~1 centimeter to 5 micrometers in width can be imaged. SEM can be used to image bacteria, viruses, tissues as well as larger samples like insects. Conventional SEM gives a magnification ranging from 20X to 30,000X and spatial resolution of 50 to 100 nanometers.
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Related Experiment Video

Updated: Mar 29, 2026

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions
13:43

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Published on: June 24, 2013

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Stochastic Micro-Pattern for Automated Correlative Fluorescence - Scanning Electron Microscopy.

Isabell Begemann1,2, Abhiyan Viplav1,2, Christiane Rasch2

  • 1DFG Cluster of Excellence 'Cells in Motion', (EXC 1003).

Scientific Reports
|December 10, 2015
PubMed
Summary
This summary is machine-generated.

We developed a rapid, low-cost method using open-source tools to align fluorescence microscopy images with scanning electron micrographs. This technique accurately combines live cell and ultrastructural data for cellular surface studies.

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Area of Science:

  • Cell Biology
  • Microscopy
  • Biophysics

Background:

  • Correlative microscopy combines live-cell fluorescence imaging with high-resolution ultrastructural data from scanning electron microscopy.
  • Current alignment methods are often complex, time-consuming, and expensive.
  • Accurate spatial correlation is crucial for understanding cellular surface features.

Purpose of the Study:

  • To develop a rapid, cost-effective, and accessible method for aligning fluorescence microscopy images with scanning electron micrographs.
  • To enable routine correlative fluorescence-scanning electron microscopy in standard laboratories.
  • To facilitate the study of protein localization at cellular ultrastructures.

Main Methods:

  • Development of a stochastic gold micro-pattern for fiducial alignment.
  • Utilizing open-source software and standard laboratory equipment.
  • Software-assisted image processing for accurate spatial registration.

Main Results:

  • Successful alignment of fluorescence signals with scanning electron micrographs using the gold micro-pattern.
  • Achieved alignment accuracy comparable to existing, more resource-intensive methods.
  • Demonstrated the method's robustness and ease of integration into existing workflows.

Conclusions:

  • The developed method offers a versatile, robust, and affordable solution for correlative fluorescence-scanning electron microscopy.
  • This approach democratizes advanced correlative imaging techniques for cellular surface studies.
  • Facilitates detailed investigation of protein localization at membrane ultrastructures.