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Correlative Fluorescence and Electron Microscopy in 3D-Scanning Electron Microscope Perspective.

Jonathan Franks1, Callen T Wallace1, Masateru Shibata2

  • 1Center for Biologic Imaging, University of Pittsburgh, Pittsburgh, Pennsylvania.

Current Protocols in Cytometry
|April 4, 2017
PubMed
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Correlating fluorescence microscopy (FM) and electron microscopy (EM) data is crucial for biological imaging. This study introduces a novel 3D EM tomogram method to directly link FM findings with high-resolution EM data.

Area of Science:

  • Biological imaging
  • Microscopy techniques
  • Cell biology

Background:

  • Correlating fluorescence microscopy (FM) and electron microscopy (EM) data is vital for bridging resolution gaps in biological specimens.
  • Previous methods were limited to 2D EM navigation or separate high-resolution TEM tomography.
  • A unified approach is needed for direct correlation of FM and EM data.

Purpose of the Study:

  • To develop a novel method for directly correlating 3D electron microscopy (EM) tomograms with prior fluorescence microscopy (FM) data.
  • To overcome limitations of existing techniques for correlative light and electron microscopy.
  • To enhance the ability to bridge the resolution gap between FM and EM.

Main Methods:

  • Embedding samples previously analyzed by FM.
Keywords:
EM tomographySEMfluorescencelow vacuummechanical polishing

Related Experiment Videos

  • Sequential mechanical polishing of embedded samples.
  • Acquiring 3D EM tomograms using scanning electron microscopy (SEM) with backscatter imaging.

    • Main Results:

    • Successful generation of 3D EM tomograms from sequentially polished samples.
    • Direct correlation of the resulting 3D EM tomograms with the initial FM data.
    • Demonstration of a novel approach for correlative FM-EM analysis.

    Conclusions:

    • The presented method enables direct correlation of 3D EM tomograms with FM data.
    • This novel approach overcomes previous limitations in correlative microscopy.
    • Facilitates a more comprehensive understanding of biological structures at multiple resolutions.