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Total internal reflection fluorescence microscopy or TIRF is an advanced microscopic technique used to visualize fluorophores in samples close to a solid surface with a higher refractive index, such as a glass coverslip. TIRF only allows fluorophores in proximity to the solid surface to be excited. When light from a medium with a lower refractive index (such as air) hits the glass coverslip at a critical angle, the light undergoes total internal reflection stead of passing through the glass.
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The wavelengths of visible light ultimately limit the maximum theoretical resolution of images created by light microscopes. Most light microscopes can only magnify 1000X, and a few can magnify up to 1500X. Electrons, like electromagnetic radiation, can behave like waves, but with wavelengths of 0.005 nm, they produce significantly greater resolution up to 0.05 nm as compared to 500 nm for visible light. An electron microscope (EM) can create a sharp image that is magnified up to 2,000,000X.
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Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions
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Customized patterned substrates for highly versatile correlative light-scanning electron microscopy.

Lorena Benedetti1, Elisa Sogne2, Simona Rodighiero3

  • 11] Fondazione Filarete for Biosciences and Innovation, Viale Ortles 22/4, 20139, Milan, Italy [2] Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano, and National Research Council (CNR) Neuroscience Institute, Via Vanvitelli 32, 20129 Milan, Italy.

Scientific Reports
|November 14, 2014
PubMed
Summary
This summary is machine-generated.

Correlative light electron microscopy (CLEM) enables nanometre resolution imaging of dynamic cellular events. Customized patterned glass substrates enhance the feasibility of fluorescence and scanning electron microscopy correlative studies.

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Area of Science:

  • Cell biology
  • Microscopy
  • Nanotechnology

Background:

  • Correlative light electron microscopy (CLEM) integrates light and electron microscopy for high-resolution cellular imaging.
  • Dynamic cellular processes require advanced microscopy techniques for observation at nanometre resolution.

Purpose of the Study:

  • To present customized patterned glass substrates designed to improve CLEM feasibility.
  • To enhance the integration of fluorescence/confocal and scanning electron microscopy.

Main Methods:

  • Development of customized patterned glass substrates for microscopy.
  • Application of substrates in correlative fluorescence/confocal and scanning electron microscopy.

Main Results:

  • The patterned glass substrates facilitate improved feasibility for CLEM.
  • Enhanced integration of fluorescence and scanning electron microscopy was achieved.

Conclusions:

  • Customized patterned glass substrates represent a valuable tool for advancing CLEM techniques.
  • This approach improves the ability to study dynamic cellular events at nanometre resolution.