David McLoskey

A new sub-nanosecond LED at 280 nm: application to protein fluorescence

We demonstrate measurement of the intrinsic fluorescence decay of a protein excited with a new and inexpensive optical source based on a light emitting diode (LED) giving 600 ps pulses at ∼280 nm. We believe this source will offer significant new capabilities for fluorescence research and development.

Monitoring sol-to-gel transitions via fluorescence lifetime determination using viscosity sensitive fluorescent probes.

The sol-to-gel transition was monitored via the use of time-resolved recording of the fluorescence emission of viscosity-sensitive probes. Two dyes were chosen for the study, water-soluble DASPMI and a hydrophobic BODIPY, and steady-state, time-resolved and time-tagged fluorescence measurements were performed. These techniques, coupled with the probes different solubility, allowed complementary fluorescence lifetime and intensity data to be obtained from the dyes introduced into the matrix-forming mixture to produce sol-gel derived monoliths. Two different precursors were used as examples. A hydrogel was formed from a commercially available gellan gum (Gelrite), and a glass-like monolith was formed using tetraethyl orthosilicate. Changes in fluorescence lifetime could be related to those in the local viscosity sensed by the probe. The combination of this type of probe with time-resolved measurements is extremely useful in monitoring the microscopic changes that occur during the sol-to-gel transition within this important class of materials.

Selective excitation of tryptophan fluorescence decay in proteins using a subnanosecond 295nm light-emitting diode and time-correlated single-photon counting

We demonstrate an AlGaN light-emitting diode (LED) giving pulses of ∼600ps full width half maximum, 0.35μW average power, 0.6mW peak power, and ∼12nm bandwidth at 295nm. This source is ideal for protein intrinsic tryptophan fluorescence decay research without the unwanted excitation of tyrosine and paves the way to lab-on-a-chip protein assays using fluorescence decay times. Fluorescence decay and anisotropy decay measurements of human serum albumin are reported and the usefulness of the 295nm LED demonstrated in comparisons with a nanosecond flashlamp and LEDs with nominal wavelength emission of 280nm.

The fluorescence and circular dichroism of proteins in reverse micelles: application to the photophysics of human serum albumin and N-acetyl-L-tryptophanamide

Evidence is presented that a compartmentalised protein exists in its native state only within a particular size of aqueous cavity. This behaviour is shown to exist in AOT reverse micelles using fluorescence quenching and circular dichroism (CD) studies of human serum albumin (HSA). In particular, far ultraviolet CD measurements show that a reduction in quencher accessibility to the fluorophore is consistent with the protein being nearest to its native conformation at a waterpool size of around 80 A diameter. We also show that the biexponential fluorescence decay of N-acetyl-L-tryptophanamide (NATA) in AOT reverse micelles arises from the probe being located in two distinct sites within the interfacial region. The more viscous of these two sites is located on the waterpool side of the interface and the other is located on the oil side of the interface.

Multiplexed single-photon counting. I. A time-correlated fluorescence lifetime camera

We report the operation of a new type of fluorescence lifetime imaging camera based on the time‐correlated single‐photon counting (TCSPC) technique. To the best of our knowledge the application‐specific integrated circuit (ASIC) used in the camera is the first ASIC designed for the field of fluorescence. The ASIC also forms the basis for the first read‐out system for single‐photon timing array detectors and is capable of multiplexing and routing counts from up to sixteen detection channels, while preserving their timing characteristics with picosecond resolution. In conjunction with an array detector such as a multianode MCP‐PM this ASIC allows multiple fluorescence decays to be routinely and simultaneously acquired using a single set of TCSPC timing electronics. To demonstrate one practical application of this technology, we have observed for the first time the spatial distribution of fluorescence lifetime contours through a strongly self‐absorbing sample, and the effects observed demonstrate how differe...

Excitation of fluorescence decay using a 265 nm pulsed light-emitting diode: Evidence for aqueous phenylalanine rotamers

The authors describe the characteristics and application of a 265nm AlGaN light-emitting diode (LED) operated at 1MHz repetition rate, 1.2ns pulse duration, 1.32μW average power, 2.3mW peak power, and ∼12nm bandwidth. The LED enables the fluorescence decay of weakly emitting phenylalanine to be measured routinely, even in dilute solution. For pH of 6–9.2, the authors find evidence for a biexponential rather than monoexponential decay, providing direct evidence for the presence of phenylalanine rotamers with a photophysics closer to the other two fluorescent amino acids tryrosine and tryptophan than has previously been reported.

Multiplexed single-photon counting. II. The statistical theory of time-correlated measurements

We present the statistical theory governing the operation of multiplexed time‐correlated single‐photon counting (MUX‐TCSPC) fluorometers which incorporate multiple detection channels and a single time‐to‐amplitude converter. The requirement to reject coincident counts in different detection channels leads to binomial photon counting statistics. The overall data collection probability is mapped out in terms of the ‘‘start’’ repetition rate, the number of detection channels, the ‘‘stop‐to‐start’’ rate ratio per detector, and the dead time. The overall collection probability is shown not to be proportional to the number of detection channels, but to be maximized at ∼0.37, which still compares very favorably with the equivalent pileup limited probability of ∼0.01 for a single detection channel. Excellent agreement with our theory is demonstrated for experimental data with 2 and 16 detection channels. Potential sources of errors when using MUX‐TCSPC to record fluorescence anisotropy and spectra are described. The theory we describe provides a model for photon counting with multiple detection channels which is quite general and also applicable to other fields such as time‐resolved imaging using photon migration in tissue and optical time‐domain reflectometry.

Multiplexed time-correlated single-photon counting.

We review the technique of multiplexed time-correlated single-photon counting whereby multiple fluorescence decay curves are recorded in parallel by statistically time-sharing a single time-to-amplitude converter. Application of the multiplexing technique to measuring the fluorescence lifetime topography of a self-absorbing sample is demonstrated. Further possibilities are discussed for multiplexed optical fiber sensor networks with built-in intelligence for detecting and discriminating between different metal ions in solution.

Fast time-correlated single-photon counting fluorescence lifetime acquisition using a 100 MHz semiconductor excitation source

The fast and efficient collection of time-resolved fluorescence decay data is shown using a high repetition rate semiconductor laser excitation source optimally matched to low dead-time counting electronics. The 100 MHz repetition rate allows a measurement range of 10 ns without re-excitation of the sample. Rapid acquisition of time-resolved fluorescence data is essential for microscopy applications, such as fluorescence lifetime imaging. With this in mind we measured a representative dye (lifetime 376 ps) to determine the fastest collection time for a single exponential decay. Other dyes were also used with lifetimes ranging from ~90 ps to 4 ns.

In situ formation of silver nanostructures within a polysaccharide film and application as a potential biocompatible fluorescence sensing medium

Simple to manufacture polysaccharide films containing a silver salt, from which silver nanostructures can be produced in situ by light irradiation, were investigated for possible biosensing applications. The silver nanostructures were patterned in situ within a film and cast from a liquid solution of gellan gum, using a compact time-resolved fluorescence microscope. The position and time of irradiation, made using a semiconductor laser in CW mode, were computer controlled. Evidence for their formation was obtained via UV-vis spectroscopy, AFM and SEM-EDAX. On drying the polysaccharide film exhibited a viscosity increase of several orders of magnitude, which was elucidated by changes in the fluorescence lifetime of a probe molecule (DASPMI). To demonstrate the potential for biocompatible sensing applications the influence of the presence of areas of silver nanostructures on the fluorescence of a protein (bovine serum albumin) labelled with fluorescein isothiocyanate was monitored via fluorescence lifetime imaging and the photophysical behaviour found to be consistent with a metal induced increase in the radiative decay rate.