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Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging
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Optimizing fluorophore density for single virus counting: a photophysical approach.

Swarupa Chatterjee1,2, Robert Molenaar1, Leroy Tromp1

  • 1Nanobiophysics (NBP), MESA + Institute for Nanotechnology and Technical Medical Centre, Faculty of Science and Technology, University of Twente, PO Box 217, 7500 AE Enschede, The Netherlands.

Methods and Applications in Fluorescence
|January 22, 2021
PubMed
Summary

Optimizing fluorescent labels on cowpea chlorotic mottle virus (CCMV) enhances particle brightness. This allows for precise quantification of low-concentration CCMV in health and environmental studies using single particle counting.

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Area of Science:

  • Biophysics
  • Nanotechnology
  • Biochemistry

Background:

  • Quantifying low concentrations of single nanoparticles is crucial for health and environmental research.
  • Fluorophore self-quenching limits nanoparticle brightness, posing challenges for accurate quantification.

Purpose of the Study:

  • To investigate how the number of fluorescent labels affects the brightness of cowpea chlorotic mottle virus (CCMV).
  • To understand the photophysical interactions between fluorophores on CCMV particles.
  • To establish a method for quantifying low-concentration CCMV using single particle counting.

Main Methods:

  • Fluorescently labelling cowpea chlorotic mottle virus (CCMV) with varying numbers of labels.
  • Analyzing the photophysical interplay between fluorophores on individual virus particles.
  • Single particle counting experiments to determine CCMV concentrations.

Main Results:

  • Fluorophore self-quenching and energy transfer to dark aggregates limit achievable particle brightness.
  • Careful selection of label numbers minimizes these negative effects, enhancing total particle brightness.
  • Successful quantification of fluorescently labelled CCMV down to femtomolar (fM) concentrations was achieved.

Conclusions:

  • Optimizing the number of fluorescent labels per CCMV particle is essential for maximizing brightness.
  • This optimized approach enables sensitive quantification of CCMV at very low concentrations.
  • The findings are applicable to health and environmental research requiring single nanoparticle quantification.