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Related Concept Videos

Immunogold Electron Microscopy01:20

Immunogold Electron Microscopy

Immunoelectron microscopy utilizes immunogold labeling of endogenous proteins with specific antibodies to detect and localize these proteins in cells and tissues. The procedure provides insights into the distribution and quantification of protein under different stimulation conditions offering clues about their functions. Conjugating highly electron-dense gold particles with primary or secondary antibodies allow antigen detection on and within cells, with high resolution and specificity.
Immunofluorescence Microscopy01:12

Immunofluorescence Microscopy

A fluorescence microscope uses fluorescent chromophores called fluorochromes, which can absorb energy from a light source and then emit this energy as visible light. Fluorochromes include naturally fluorescent substances (such as chlorophylls) and fluorescent stains that are added to the specimen to create contrast. Dyes such as Texas red and FITC are examples of fluorochromes. Other examples include the nucleic acid dyes 4’,6’-diamidino-2-phenylindole (DAPI), and acridine orange.
The...
Super-resolution Fluorescence Microscopy01:37

Super-resolution Fluorescence Microscopy

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 developed.

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Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles
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Optimizing immuno-labeling for correlative fluorescence and electron microscopy on a single specimen.

Matthia A Karreman1, Alexandra V Agronskaia, Elly G van Donselaar

  • 1Molecular Biophysics, Department of Physics and Astronomy, Utrecht University, Princetonplein 1, Utrecht, The Netherlands. M.A.Karreman@uu.nl

Journal of Structural Biology
|September 18, 2012
PubMed
Summary

Correlative microscopy requires careful sample preparation. This study compares fluorescent probe performance and presents a novel protocol for combined fluorescence and electron microscopy.

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Area of Science:

  • Cell biology
  • Microscopy techniques

Background:

  • Correlative fluorescence and electron microscopy (iLEM) integrates FM and TEM for high-resolution imaging.
  • Sample preparation is critical for dual FM and TEM imaging of single specimens.
  • Fluorescent probe performance can be affected by the TEM's vacuum environment.

Purpose of the Study:

  • To compare the fluorescence intensity of six probes in dry, oxygen-free conditions versus aqueous environments.
  • To develop and validate a single specimen preparation protocol for iLEM.

Main Methods:

  • Comparative analysis of six fluorescent probes under varying environmental conditions (dry/oxygen-free vs. water).
  • Development of a freeze-substitution and Lowicryl resin embedding protocol.
  • Evaluation of the protocol's suitability for both fluorescence microscopy (FM) and transmission electron microscopy (TEM).

Main Results:

  • Fluorescence intensity of certain probes is significantly influenced by their surrounding environment.
  • The developed freeze-substitution and Lowicryl embedding protocol preserves fluorescent immuno-labeling.
  • The protocol provides excellent membrane contrast for TEM imaging.

Conclusions:

  • Environmental factors critically impact fluorophore performance in correlative microscopy.
  • The presented single-specimen preparation protocol is effective for iLEM, ensuring good FM and TEM results.
  • This protocol is valuable for various correlative microscopy applications.