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Imaging of Isolated Extracellular Vesicles Using Fluorescence Microscopy.

Dmitry Ter-Ovanesyan1,2,3,4, Emma J K Kowal1,3, Aviv Regev4,5

  • 1Department of Genetics, Harvard Medical School, Boston, MA, USA.

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|August 23, 2017
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Summary

High-resolution microscopy reveals hidden heterogeneity in extracellular vesicles (EVs). This protocol uses fluorescent staining and TIRF-M for detailed single EV analysis, overcoming bulk analysis limitations.

Keywords:
EVsExosomesExtracellular vesiclesImagingMicroscopyTIRF

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Area of Science:

  • Biophysics
  • Cell Biology
  • Biochemistry

Background:

  • High-resolution fluorescence microscopy allows single-object studies, revealing heterogeneity missed by bulk analysis.
  • Extracellular vesicles (EVs) are crucial in intercellular communication, but their heterogeneity is often masked.
  • Single-object analysis offers a powerful approach to characterize distinct EV subpopulations.

Purpose of the Study:

  • To present a protocol for analyzing extracellular vesicles (EVs) at the single-object level.
  • To enable the characterization of specific EV subsets using high-resolution microscopy.
  • To overcome limitations of ensemble averaging in conventional biochemical analysis of EVs.

Main Methods:

  • Isolation of extracellular vesicles (EVs).
  • Staining of isolated EVs with a fluorescent lipid dye.
  • Attachment of stained EVs onto glass slides for imaging.
  • Utilizing high-resolution microscopy techniques, specifically total internal reflection fluorescence microscopy (TIRF-M).

Main Results:

  • A detailed protocol for preparing EVs for high-resolution imaging is established.
  • The method allows for the visualization and analysis of individual EVs.
  • Potential to uncover previously undetectable heterogeneity within EV populations.

Conclusions:

  • Single-object analysis of EVs using high-resolution microscopy is feasible and advantageous.
  • The presented protocol facilitates detailed characterization of EV subsets.
  • This approach enhances our understanding of EV heterogeneity and function.