Open AccessDark-Field Illumination on Zero-Mode Waveguide/Microfluidic Hybrid Chip Reveals T4 Replisomal Protein Interactions
Author(s) -
Yanhui Zhao,
Danqi Chen,
Hongjun Yue,
Michelle M. Spiering,
Chenglong Zhao,
Stephen J. Benkovic,
Tony Jun Huang
Publication year - 2014
Publication title -
nano letters
Language(s) - Uncategorized
Resource type - Journals
SCImago Journal Rank - 4.853
H-Index - 488
eISSN - 1530-6992
pISSN - 1530-6984
DOI - 10.1021/nl404802f
Subject(s) - biomolecule , fluorescence , microscopy , fluorescence microscope , förster resonance energy transfer , microfluidics , materials science , fluorescence lifetime imaging microscopy , optics , optoelectronics , chemistry , nanotechnology , physics
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.