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Related Concept Videos

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Conducting Multiple Imaging Modes with One Fluorescence Microscope
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Integrated super resolution fluorescence microscopy and transmission electron microscopy.

Sajjad Mohammadian1, Alexandra V Agronskaia1, Gerhard A Blab1

  • 1Molecular Biophysics, Department of Physics, Faculty of Science, Utrecht University, Princetonplein 1, 3584 CC Utrecht, Netherlands.

Ultramicroscopy
|May 30, 2020
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Summary
This summary is machine-generated.

This study presents an integrated super-resolution fluorescence microscopy (FM) and transmission electron microscopy (TEM) system. This novel approach enhances correlative light and electron microscopy (CLEM) by reducing resolution gaps and improving sample handling.

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Area of Science:

  • Microscopy
  • Cell Biology
  • Biophysics

Background:

  • Correlative light and electron microscopy (CLEM) combines fluorescence microscopy (FM) and electron microscopy (EM) for multi-modal imaging.
  • A key limitation in CLEM is the significant resolution difference between FM and EM.
  • Current CLEM workflows often involve separate setups, risking sample damage and complicating region identification.

Purpose of the Study:

  • To develop and present an integrated super-resolution FM and TEM system for enhanced CLEM.
  • To reduce the resolution gap between FM and EM in CLEM.
  • To improve correlation accuracy and streamline the CLEM workflow by avoiding sample transfer.

Main Methods:

  • Integration of a wide-field super-resolution FM within a Transmission Electron Microscope (TEM).
  • Utilized a rotating TEM sample holder for seamless switching between imaging modalities.
  • Employed fiducial markers for accurate image overlay between FM and TEM datasets.
  • Experimentally validated on Lowicryl embedded Human Umbilical Vein Endothelial Cells labeled for Caveolin.

Main Results:

  • Demonstrated successful overlay of TEM and FM images with an accuracy of 28 ± 11 nm.
  • Achieved overlay accuracy close to the optical resolution limit of ~50 nm.
  • Presented initial imaging experiments on labeled endothelial cells, showcasing the system's capability.

Conclusions:

  • The integrated super-resolution CLEM system effectively bridges the resolution gap between FM and EM.
  • The presented approach minimizes sample manipulation, reducing potential damage and improving workflow efficiency.
  • This integrated system offers a promising advancement for high-resolution cellular ultrastructure analysis in correlative microscopy.