Dark-field illumination on zero-mode waveguide/microfluidic hybrid chip reveals T4 replisomal protein interactions

Yanhui Zhao, Danqi Chen, Hongjun Yue, Michelle M. Spiering, Chenglong Zhao, Stephen J. Benkovic, Tony Jun Huang

Research output: Contribution to journalArticlepeer-review

29 Scopus citations


The ability of zero-mode waveguides (ZMWs) to guide light energy into subwavelength-diameter cylindrical nanoapertures has been exploited for single-molecule fluorescence studies of biomolecules at micromolar concentrations, the typical dissociation constants for biomolecular interactions. Although epi-fluorescence microscopy is now adopted for ZMW-based imaging as an alternative to the commercialized ZMW imaging platform, its suitability and performance awaits rigorous examination. Here, we present conical lens-based dark-field fluorescence microscopy in combination with a ZMW/microfluidic chip for single-molecule fluorescence imaging. We demonstrate that compared to epi-illumination, the dark-field configuration displayed diminished background and noise and enhanced signal-to-noise ratios. This signal-to-noise ratio for imaging using the dark-field setup remains essentially unperturbed by the presence of background fluorescent molecules at micromolar concentration. Our design allowed single-molecule FRET studies that revealed weak DNA-protein and protein-protein interactions found with T4 replisomal proteins.

Original languageEnglish (US)
Pages (from-to)1952-1960
Number of pages9
JournalNano letters
Issue number4
StatePublished - Apr 9 2014

All Science Journal Classification (ASJC) codes

  • Bioengineering
  • General Chemistry
  • General Materials Science
  • Condensed Matter Physics
  • Mechanical Engineering


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