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Updated: May 6, 2026

Author Spotlight: Advancements in Correlative Light and Electron Microscopy with Fluorescent Protein Preservation
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Correlative photoactivated localization and scanning electron microscopy.

Benjamin G Kopek1, Gleb Shtengel, Jonathan B Grimm

  • 1Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, United States of America.

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|November 9, 2013
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Researchers developed a simpler correlative microscopy technique. This method combines 2D Photoactivated Localization Microscopy (PALM) with scanning electron microscopy (SEM) for precise protein localization and ultrastructure preservation, making advanced cell biology more accessible.

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Area of Science:

  • Cell Biology
  • Microscopy
  • Biophysics

Background:

  • Precise subcellular protein localization is vital for understanding biological systems.
  • Previous correlative microscopy methods achieved high resolution but required specialized equipment, limiting accessibility.
  • Simultaneous preservation of fluorophores and ultrastructure is challenging.

Purpose of the Study:

  • To present a faster, more practical correlative microscopy protocol.
  • To enable high-resolution protein localization and ultrastructure preservation using simpler instrumentation.
  • To expand the accessibility of advanced correlative microscopy techniques.

Main Methods:

  • Developed a modified Tokuyasu cryosection staining protocol for standard scanning electron microscopy (SEM).
  • Utilized two-dimensional Photoactivated Localization Microscopy (PALM) for fluorescent protein localization.
  • Correlated PALM images with SEM ultrastructure without focused ion beam ablation.
  • Demonstrated labeling of mitochondrial nucleoids, peroxisomes, nuclear lamina, and actin.

Main Results:

  • Achieved simultaneous fluorophore and ultrastructure preservation using a simplified PALM-SEM system.
  • Successfully labeled diverse cellular compartments and structures.
  • Demonstrated simultaneous two-color PALM imaging correlated with electron micrographs.
  • Showcased versatility with small-molecule dyes for actin labeling.

Conclusions:

  • The new protocol simplifies correlative microscopy tools while maintaining high-resolution protein labeling and ultrastructural detail.
  • This accessible technique is valuable for a wide range of researchers studying cellular organization and function.
  • Expanded labeling options enhance the utility of correlative super-resolution microscopy.