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Conducting Multiple Imaging Modes with One Fluorescence Microscope
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Retarding Field Integrated Fluorescence and Electron Microscope.

Yoram Vos1, Ryan I Lane1, Chris J Peddie2

  • 1Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, Delft2628CJ, The Netherlands.

Microscopy and Microanalysis : the Official Journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada
|December 22, 2020
PubMed
Summary
This summary is machine-generated.

A new retarding field in integrated fluorescence and electron microscopy enhances signal strength for delicate samples. This technique improves imaging for ultra-thin sections and enables new studies on electron beam damage.

Keywords:
SEMbackscattered electron detectioncorrelative light and electron microscopyelectron beam induced damageretarding field

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Area of Science:

  • Correlative Light and Electron Microscopy
  • Materials Science
  • Biophysics

Background:

  • Integrated fluorescence and electron microscopy (iFEM) requires delicate sample preparation to preserve fluorescence.
  • Conventional electron microscopy (EM) sample preparation often uses high staining, which can damage fluorescence.
  • Optimizing signal collection in iFEM is crucial for high-resolution imaging of biological and material samples.

Purpose of the Study:

  • To introduce and evaluate a retarding field system for iFEM.
  • To demonstrate signal enhancement in EM imaging of iFEM samples.
  • To explore the application of low-energy electron imaging for assessing beam-induced damage.

Main Methods:

  • Implementation of a retarding field between the electron objective lens and sample in an iFEM.
  • Testing the retarding field on 80-nm immunolabeled sections and 100-nm in-resin fluorescence sections.
  • Optimizing electron landing energy and retarding field parameters for ultra-thin (50 nm) sections.
  • Utilizing the system for low-energy electron imaging (down to a few eV) with high energy dispersion (0.3 eV).

Main Results:

  • The retarding field significantly enhanced signal collection and strength in the electron microscope for iFEM samples.
  • Signal improvement was demonstrated for both post-embedding immunolabeled and in-resin fluorescence sections.
  • Tuning the retarding field and landing energy optimized imaging for ultra-thin sections.
  • The system achieved low landing energies, enabling in situ quantification of fluorescence bleaching due to electron beam irradiation.

Conclusions:

  • The integrated retarding field is an effective method for improving EM signal in iFEM, especially for samples with reduced staining.
  • This technique facilitates high-quality imaging of delicate specimens and ultra-thin sections.
  • The ability to perform low-energy electron imaging opens new avenues for studying electron beam-induced sample damage in situ.