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Related Experiment Video

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Nanoparticle Tracking Analysis for the Quantification and Size Determination of Extracellular Vesicles
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High throughput inclusion body sizing: Nano particle tracking analysis.

Wieland N Reichelt1, Andreas Kaineder1, Markus Brillmann1

  • 1Christian Doppler Laboratory for Mechanistic and Physiological Methods for Improved Bioprocesses, Institute of Chemical Engineering, Vienna University of Technology, Vienna, Austria.

Biotechnology Journal
|March 17, 2017
PubMed
Summary

This study introduces improved methods for quantifying protein aggregation in inclusion bodies (IBs) within cells. Transmission electron microscopy and nanoparticle tracking analysis offer more accurate and representative measurements of IB size and properties.

Keywords:
Electron microscopyGrey scale image segmentationInclusion bodyNano particle tracking analysisStickiness

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Area of Science:

  • Biotechnology
  • Cell Biology
  • Protein Science

Background:

  • Protein aggregation in Escherichia coli often leads to inclusion body (IB) formation, impacting pharmaceutical protein production.
  • Existing methods for quantifying IB size lack sensitivity and orthogonal verification.

Purpose of the Study:

  • To establish reliable and high-throughput methods for quantifying inclusion body size and physical properties.
  • To compare the efficacy of transmission electron microscopy (TEM) with nanoparticle tracking analysis (NTA) for IB characterization.

Main Methods:

  • High pressure freezing and automated freeze substitution for TEM to preserve cellular structure.
  • Manual grey scale image segmentation of TEM images for quantifying IB area.
  • Nanoparticle tracking analysis (NTA) for measuring IB diameter based on Brownian motion in cell homogenates.

Main Results:

  • TEM imaging with image segmentation provides an orthogonal method for sizing cytosolic inclusion bodies.
  • NTA, despite potential alteration of native shape by homogenization, offers a fast, high-throughput particle-based analysis.
  • The ratio of particle counts in non-fixated versus fixated samples may indicate particle stickiness.

Conclusions:

  • Image segmentation of TEM images serves as a validated orthogonal method for biological particle sizing.
  • NTA is established as a superior, high-throughput method for accurate and representative analysis of inclusion bodies compared to existing techniques.