Mitigating unwanted photophysical processes for improved single-molecule fluorescence imaging. Academic Article uri icon

Overview

MeSH

  • Benzyl Alcohols
  • Carbocyanines
  • Chromans
  • Cyclic AMP
  • Fluorescence
  • Kinetics
  • Photobleaching

MeSH Major

  • Fluorescence Resonance Energy Transfer
  • Oligodeoxyribonucleotides
  • Ribosomes

abstract

  • Organic fluorophores common to fluorescence-based investigations suffer from unwanted photophysical properties, including blinking and photobleaching, which limit their overall experimental performance. Methods to control such processes are particularly important for single-molecule fluorescence and fluorescence resonance energy transfer imaging where uninterrupted, stable fluorescence is paramount. Fluorescence and FRET-based assays have been carried out on dye-labeled DNA and RNA-based systems to quantify the effect of including small-molecule solution additives on the fluorescence and FRET behaviors of both cyanine and Alexa fluorophores. A detailed dwell time analysis of the fluorescence and FRET trajectories of more than 200,000 individual molecules showed that two compounds identified previously as triplet state quenchers, cyclooctatetraene, and Trolox, as well as 4-nitrobenzyl alcohol, act to favorably attenuate blinking, photobleaching, and influence the rate of photoresurrection in a concentration-dependent and context-dependent manner. In both biochemical systems examined, a unique cocktail of compounds was shown to be optimal for imaging performance. By simultaneously providing the most rapid and direct access to multiple photophysical kinetic parameters, smFRET imaging provides a powerful avenue for future investigations aimed at discovering new compounds, and effective combinations thereof. These efforts may ultimately facilitate tuning organic dye molecule performance according to each specific experimental demand.

publication date

  • March 18, 2009

has subject area

  • Benzyl Alcohols
  • Carbocyanines
  • Chromans
  • Cyclic AMP
  • Fluorescence
  • Fluorescence Resonance Energy Transfer
  • Kinetics
  • Oligodeoxyribonucleotides
  • Photobleaching
  • Ribosomes

Research

keywords

  • Journal Article

Identity

Language

  • eng

PubMed Central ID

  • PMC2907709

Digital Object Identifier (DOI)

  • 10.1016/j.bpj.2008.11.061

PubMed ID

  • 19289062

Additional Document Info

start page

  • 2371

end page

  • 2381

volume

  • 96

number

  • 6