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Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy
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Fluorescence lifetime imaging and electron microscopy: a correlative approach.

Johannes G Wieland1, Nilanjon Naskar2, Angelika Rück2

  • 1Central Facility for Electron Microscopy, Ulm University, 89081, Ulm, Germany. johannes.wieland@uni-ulm.de.

Histochemistry and Cell Biology
|March 10, 2022
PubMed
Summary

This study introduces a novel correlative workflow combining fluorescence lifetime imaging microscopy (FLIM) and electron microscopy (EM) to analyze cellular metabolism. The method reveals metabolic changes and structural alterations in cells, offering new insights into diseases.

Keywords:
Cell metabolismCorrelative light- and electron microscopy (CLEM)Fluorescence lifetime imaging and electron microscopy (FLEM)Fluorescence lifetime imaging microscopy (FLIM)NADHSTEM tomography

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Area of Science:

  • Cellular and Molecular Biology
  • Biophysics
  • Microscopy Techniques

Background:

  • Cellular metabolism is crucial for understanding various pathophysiological processes.
  • Fluorescence lifetime imaging microscopy (FLIM) quantifies metabolic cofactors like nicotinamide adenine dinucleotide hydrogen (NADH).
  • Electron microscopy (EM) offers high spatial resolution for ultrastructural analysis.

Purpose of the Study:

  • To develop and validate a correlative imaging workflow integrating FLIM and EM.
  • To investigate cellular metabolic changes and ultrastructural alterations simultaneously.
  • To explore the potential of this combined approach for studying diseases like cancer and neurodegenerative disorders.

Main Methods:

  • Human fibroblasts were cultured on a coordinate microscopy slide for precise sample navigation.
  • Fluorescence lifetime imaging microscopy (FLIM) was performed to measure NADH lifetime.
  • Cells were chemically fixed, embedded, sectioned, and imaged using scanning transmission electron microscopy (STEM) tomography.

Main Results:

  • Correlative imaging of antimycin A-treated fibroblasts demonstrated a decrease in NADH fluorescence lifetime.
  • STEM tomography revealed associated mitochondrial swelling and large cavities in treated cells.
  • This marks the first successful integration of FLIM and EM for correlative cellular analysis.

Conclusions:

  • The correlative FLIM-EM workflow provides a powerful tool for investigating cellular metabolism and structure.
  • This integrated approach enhances the study of metabolic dysfunctions in diseases.
  • The technique holds significant promise for advancing research in cancer, neurodegenerative disorders, and viral infections.