Optimizing fluorophore density for single virus counting: A photophysical approach

Swarupa Chatterjee, Robert Molenaar, Leroy Tromp, R. Martijn Wagterveld, Hendrik D.W. Roesink, Jeroen J.L.M. Cornelissen, Mireille M.A.E. Claessens, Christian Blum*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

8 Citations (Scopus)
120 Downloads (Pure)


In health and environmental research, it is often necessary to quantify the concentrations of single (bio) nanoparticles present at very low concentrations. Suitable quantification approaches that rely on counting and tracking of single fluorescently labelled (bio) nanoparticles are often challenging since fluorophore self-quenching limits the maximum particle brightness. Here we study how the number of labels per nanoparticle influences the total brightness of fluorescently labelled cowpea chlorotic mottle virus(CCMV). We analyze in detail the photophysical interplay between the fluorophores on the virus particles. We deduce that the formation of dark aggregates and energy transfer towards these aggregates limits the total particle brightness that can be achieved. We show that by carefully selecting the number of fluorescent labels per CCMV, and thus minimizing the negative effects on particle brightness, it is possible to quantify fluorescently labelled CCMV concentrations down to fM concentrations in single particle counting experiments.

Original languageEnglish
Article number025001
JournalMethods and Applications in Fluorescence
Issue number2
Publication statusPublished - 20 Jan 2021


  • UT-Hybrid-D
  • Low concentration
  • Self quenching
  • Single particle detection
  • Virus
  • H-aggregates
  • self quenching
  • single particle detection
  • h-aggregates
  • low concentration
  • virus


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