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Related Concept Videos

Overview of Electron Microscopy01:25

Overview of Electron Microscopy

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The wavelengths of visible light ultimately limit the maximum theoretical resolution of images created by light microscopes. Most light microscopes can only magnify 1000X, and a few can magnify up to 1500X. Electrons, like electromagnetic radiation, can behave like waves, but with wavelengths of 0.005 nm, they produce significantly greater resolution up to 0.05 nm as compared to 500 nm for visible light. An electron microscope (EM) can create a sharp image that is magnified up to 2,000,000X.
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Immunoelectron microscopy utilizes immunogold labeling of endogenous proteins with specific antibodies to detect and localize these proteins in cells and tissues. The procedure provides insights into the distribution and quantification of protein under different stimulation conditions offering clues about their functions. Conjugating highly electron-dense gold particles with primary or secondary antibodies allow antigen detection on and within cells, with high resolution and specificity.
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Updated: May 30, 2025

Digital Droplet PCR Method for the Quantification of AAV Transduction Efficiency in Murine Retina
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Low voltage electron microscopy: An emerging tool for AAV characterization.

Kevin D Ausman1, Neal Whitaker2, Madhumitha Balasubramanian2

  • 1Spark Therapeutics, Inc., 3025 Market Street, Philadelphia, PA 19104, United States.

Journal of Pharmaceutical Sciences
|January 30, 2025
PubMed
Summary
This summary is machine-generated.

Low voltage electron microscopy offers a cost-effective alternative for characterizing adeno-associated virus (AAV) gene therapy products. This method provides accurate empty/full analysis and insights into capsid integrity, making it accessible for more researchers.

Keywords:
Adeno-associated virus (AAV)AgglomerationBiopharmaceutical characterizationDNA deliveryGene therapyGene vector(s)Image AnalysisImaging method(s)Physical characterizationViral vector(s)

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Area of Science:

  • Biotechnology
  • Microscopy
  • Gene Therapy

Background:

  • Transmission electron microscopy (TEM) is crucial for characterizing adeno-associated virus (AAV) gene therapy products.
  • High costs and expertise requirements limit access to traditional TEM systems for many companies.
  • A need exists for more accessible and affordable characterization methods in AAV gene therapy.

Purpose of the Study:

  • To evaluate the utility of low voltage electron microscopy (LVEM) for AAV characterization.
  • To demonstrate LVEM's effectiveness in analyzing empty/full capsids, incomplete capsids, and aggregates.
  • To compare LVEM results with traditional TEM and other industry-standard methods.

Main Methods:

  • Utilized low voltage electron microscopy (LVEM) for AAV sample analysis.
  • Performed empty/full capsid ratio determination using LVEM.
  • Investigated broken/incomplete capsid morphologies and aggregation phenomena.
  • Compared LVEM findings with orthogonal methods and traditional TEM.

Main Results:

  • LVEM empty/full analysis results showed strong agreement with established orthogonal methods.
  • LVEM provided insights into the mechanism of incomplete capsid aggregation, suggesting edge passivation.
  • LVEM demonstrated capability in characterizing various AAV attributes, including aggregates and high-molecular-weight species.
  • A comprehensive analytical framework for AAV preparations using LVEM was successfully established.

Conclusions:

  • LVEM is a viable, cost-effective alternative to traditional TEM for AAV characterization.
  • LVEM facilitates detailed analysis of AAV capsid integrity and product heterogeneity.
  • The established LVEM methodology supports robust quality control for AAV gene therapy products.