Lloyd M. Davis

Single photon avalanche diode for single molecule detection

A commercially available single photon avalanche photodiode in a passively quenched circuit is used with time‐correlated single photon counting modules to achieve subnanosecond time response together with high quantum efficiency and low dark noise. These characteristics are required for experiments in single molecule detection and spectroscopy in which time‐gated detection schemes are used. By tightly focusing the input light onto the active area, a quantum efficiency of over 50% and a single photon timing jitter of 168 ps full width at half‐maximum are achieved. In addition, the full width at one‐hundredth maximum, which is of greater importance for time gating, is 790 ps, comparable to that from a microchannel plate photomultiplier. Measurements of the detector dead time, and the quantum efficiency, dark counts, time response, and pulse height distribution for different operating conditions are also reported.

THE PHOTOPHYSICAL CONSTANTS OF SEVERAL FLUORESCENT DYES PERTAINING TO ULTRASENSITIVE FLUORESCENCE SPECTROSCOPY

Abstract— The successful implementation of ultrasensitive fluorescence spectroscopy of biological and chemical species depends upon certain photophysical parameters associated with the fluorescent dye used in the investigation. These parameters include the fluorescence quantum efficiency, photodestruction quantum efficiency, absorption cross section and fluorescence lifetime. These photophysical constants were measured for several fluorescent dyes that are used for the tagging of biological species. Three different solvents, ethanol, water and a cationic surfactant used above its critical micelle concentration, were studied. The effective photon yield (ratio of the fluorescence quantum yield to the photodestruction quantum efficiency) for the dyes is nearly 100 times greater in ethanol than it is in water because of the superior photostabilities of these dyes in ethanol solvents. The implications of these parameters for the design of an ultrasensitive fluorescence experiment are discussed.

Detection and identification of single molecules in solution

We have extended our recent experiments in the detection of single fluorescent molecules in solution to the exploration of spectroscopy at the single-molecule level. As a first step we have developed a technique that can efficiently distinguish between two species of dye molecules on the basis of differences in their emission spectra. We have also demonstrated that another spectroscopic property, fluorescence lifetime, can be accurately determined at the single-molecule level. Spectroscopic properties can be used to identify fluorescent molecules and to reveal static or slowly varying aspects of the microenvironment of each molecule, thereby yielding information unavailable from bulk studies.

Rapid DNA sequencing based upon single molecule detection

We are developing a laser-based technique for the rapid sequencing of 40-kb or larger fragments of DNA at a rate of 100 to 1000 bases per second. The approach relies on fluorescent labeling of the bases in a single fragment of DNA, attachment of this labeled DNA fragment to a support, movement of the supported DNA fragment into a flowing sample stream, and detection of individual fluorescently labeled bases as they are cleaved from the DNA fragment by an exonuclease. The ability to sequence large fragments of DNA will significantly reduce the amount of subcloning and the number of overlapping sequences required to assemble megabase segments of sequence information.

Single-pulse ultrafast-laser machining of high aspect nano-holes at the surface of SiO 2

Use of high numerical aperture focusing with negative longitudinal spherical aberration is shown to enable deep (> microm), high aspect ratio, nano-scale-width holes to be machined into the surface of a fused-silica (SiO(2)) substrate with single pulses from a 200 fs, 4 microJ Ti-Sapphire laser source. The depths of the nano-holes are characterized by use of a non-destructive acetate replication technique and are confirmed by imaging of sectioned samples with a dual focused ion beam/scanning electron microscope.

Actively quenched single‐photon avalanche diode for high repetition rate time‐gated photon counting

This paper reports an experimental characterization of the EG&G SPCM‐AQ single‐photon avalanche diode module with an active quenching bias circuit that gives a dead time of ∼35 ns for use in high count rate, fast timing applications. A quantum efficiency of ≳70% and an optimal timing jitter with a full width at one‐thousandth maximum of 1.5 ns is obtained if the light is tightly focused to a spot of <25 μm in the center of the active region, if the signal from the diode before the active quenching circuitry is used as input to the timing electronics, and if an external dead time of ∼55 ns is imposed. Under these conditions, the total probability of obtaining an afterpulse is found to be ∼2×10−3. Limitations of existing time‐correlated single‐photon counting instrumentation for count rates exceeding 106 s−1 are discussed.

Ligase Detection Reaction Generation of Reverse Molecular Beacons for Near Real-Time Analysis of Bacterial Pathogens Using Single-Pair Fluorescence Resonance Energy Transfer and a Cyclic Olefin Copolymer Microfluidic Chip

Detection of pathogenic bacteria and viruses require strategies that can signal the presence of these targets in near real-time due to the potential threats created by rapid dissemination into water and/or food supplies. In this paper, we report an innovative strategy that can rapidly detect bacterial pathogens using reporter sequences found in their genome without requiring polymerase chain reaction (PCR). A pair of strain-specific primers was designed based on the 16S rRNA gene and were end-labeled with a donor (Cy5) or acceptor (Cy5.5) dye. In the presence of the target bacterium, the primers were joined using a ligase detection reaction (LDR) only when the primers were completely complementary to the target sequence to form a reverse molecular beacon (rMB), thus bringing Cy5 (donor) and Cy5.5 (acceptor) into close proximity to allow fluorescence resonance energy transfer (FRET) to occur. These rMBs were subsequently analyzed using single-molecule detection of the FRET pairs (single-pair FRET; spFRET). The LDR was performed using a continuous flow thermal cycling process configured in a cyclic olefin copolymer (COC) microfluidic device using either 2 or 20 thermal cycles. Single-molecule photon bursts from the resulting rMBs were detected on-chip and registered using a simple laser-induced fluorescence (LIF) instrument. The spFRET signatures from the target pathogens were reported in as little as 2.6 min using spFRET.

Single-nanocrystal spectroscopy of white-light-emitting CdSe nanocrystals.

We report the observation of broad-spectrum fluorescence from single CdSe nanocrystals. Individual semiconductor nanocrystals typically have a narrower emission spectrum than that of an ensemble. However, our experiments show that the ensemble white-light emission observed in ultrasmall CdSe nanocrystals is the result of many single CdSe nanocrystals, each emitting over the entire visible spectrum. These results indicate that each white-light-emitting CdSe nanocrystal contains all the trap states that give rise to the observed white-light emission.

Correlation of atomic structure and photoluminescence of the same quantum dot: pinpointing surface and internal defects that inhibit photoluminescence.

In a size regime where every atom counts, rational design and synthesis of optimal nanostructures demands direct interrogation of the effects of structural divergence of individuals on the ensemble-averaged property. To this end, we have explored the structure-function relationship of single quantum dots (QDs) via precise observation of the impact of atomic arrangement on QD fluorescence. Utilizing wide-field fluorescence microscopy and atomic number contrast scanning transmission electron microscopy (Z-STEM), we have achieved correlation of photoluminescence (PL) data and atomic-level structural information from individual colloidal QDs. This investigation of CdSe/CdS core/shell QDs has enabled exploration of the fine structural factors necessary to control QD PL. Additionally, we have identified specific morphological and structural anomalies, in the form of internal and surface defects, that consistently vitiate QD PL.

Rate equation simulation of a synchronously pumped dye laser

A simple rate equation model of a standing wave synchronously pumped dye laser yields output pulses which agree qualitatively and quantitatively with recent experimental observations. The shape, amplitude and temporal position of the simulated pulse varies dramatically, not only with cavity length detuning, but also with the gain to loss ratio. Features of pulse formation and stability are predicted which are precluded by the steady-state assumption present in most other models.