Abstract
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 language | English (US) |
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Pages (from-to) | 1952-1960 |
Number of pages | 9 |
Journal | Nano letters |
Volume | 14 |
Issue number | 4 |
DOIs | |
State | Published - Apr 9 2014 |
All Science Journal Classification (ASJC) codes
- Bioengineering
- General Chemistry
- General Materials Science
- Condensed Matter Physics
- Mechanical Engineering