Austin B. Tomaney

Real-Time DNA Sequencing from Single Polymerase Molecules

We present single-molecule, real-time sequencing data obtained from a DNA polymerase performing uninterrupted template-directed synthesis using four distinguishable fluorescently labeled deoxyribonucleoside triphosphates (dNTPs). We detected the temporal order of their enzymatic incorporation into a growing DNA strand with zero-mode waveguide nanostructure arrays, which provide optical observation volume confinement and enable parallel, simultaneous detection of thousands of single-molecule sequencing reactions. Conjugation of fluorophores to the terminal phosphate moiety of the dNTPs allows continuous observation of DNA synthesis over thousands of bases without steric hindrance. The data report directly on polymerase dynamics, revealing distinct polymerization states and pause sites corresponding to DNA secondary structure. Sequence data were aligned with the known reference sequence to assay biophysical parameters of polymerization for each template position. Consensus sequences were generated from the single-molecule reads at 15-fold coverage, showing a median accuracy of 99.3%, with no systematic error beyond fluorophore-dependent error rates.

Parallel confocal detection of single molecules in real time

The confocal detection principle is extended to a highly parallel optical system that continuously analyzes thousands of concurrent sample locations. This is achieved through the use of a holographic laser illumination multiplexer combined with a confocal pinhole array before a prism dispersive element used to provide spectroscopic information from each confocal volume. The system is demonstrated to detect and identify single fluorescent molecules from each of several thousand independent confocal volumes in real time.

Enhancement of single molecule fluorescence using conical micromirrors

Subwavelength metal apertures significantly enhance single molecule fluorescence signaling systems, but require efficient illumination and collection optics. On-chip micromirror structures offer a way to markedly improve the coupling efficiency between such subwavelength metal apertures and the external fluorescence illumination and collection optics, which in turn greatly simplifies several aspects of instrument design including optics, optomechanics, and thermal control. Modeling and experimental verification of the gains in illumination and collection efficiency for subwavelength metal apertures leads to a micromirror design that is both highly efficient yet also manufacturable. A combination of ray-based and finite-difference-time-domain models is used to optimize conical micromirrors colocated with subwavelength metal apertures for the case where the illumination light interacts strongly with the micromirror and the collection optics have modest numerical aperture (NA~0.5). Experimental methods employing either freely diffusing or immobilized dye molecules are used to measure the illumination and collection efficiencies of fabricated micromirror prototypes. An overall fluorescence gain of ~100x, comprising a 20x improvement with flood illumination efficiency together with a 5x improvement in collection efficiency, are both predicted and experimentally verified.

Integrated photonic structures for efficient, massively-parallel collection of directional single-molecule fluorescence

A conical micromirror is shown to increase the signal from fluorescent molecules within a collocated subwavelength metal aperture by 5×. Large-scale integration (75,000 miromirrors/aperture pairs) is demonstrated.