David J. S. Birch

One- and Two-Photon Excited Fluorescence Lifetimes and Anisotropy Decays of Green Fluorescent Proteins

We have used one- (OPE) and two-photon (TPE) excitation with time-correlated single-photon counting techniques to determine time-resolved fluorescence intensity and anisotropy decays of the wild-type Green Fluorescent Protein (GFP) and two red-shifted mutants, S65T-GFP and RSGFP. WT-GFP and S65T-GFP exhibited a predominant approximately 3 ns monoexponential fluorescence decay, whereas for RSGFP the main lifetimes were approximately 1.1 ns (main component) and approximately 3.3 ns. The anisotropy decay of WT-GFP and S65T-GFP was also monoexponential (global rotational correlation time of 16 +/- 1 ns). The approximately 1.1 ns lifetime of RSGFP was associated with a faster rotational depolarization, evaluated as an additional approximately 13 ns component. This feature we attribute tentatively to a greater rotational freedom of the anionic chromophore. With OPE, the initial anisotropy was close to the theoretical limit of 0.4; with TPE it was higher, approaching the TPE theoretical limit of 0.57 for the colinear case. The measured power dependence of the fluorescence signals provided direct evidence for TPE. The general independence of fluorescence decay times, rotation correlation times, and steady-state emission spectra on the excitation mode indicates that the fluorescence originated from the same distinct excited singlet states (A*, I*, B*). However, we observed a relative enhancement of blue fluorescence peaked at approximately 440 nm for TPE compared to OPE, indicating different relative excitation efficiencies. We infer that the two lifetimes of RSGFP represent the deactivation of two substates of the deprotonated intermediate (I*), distinguished by their origin (i.e., from A* or B*) and by nonradiative decay rates reflecting different internal environments of the excited-state chromophore.

Nanomedicine and its potential in diabetes research and practice

Nanomedicine involves measurement and therapy at the level of 1–100 nm. Although the science is still in its infancy, it has major potential applications in diabetes. These include solving needs such as non‐invasive glucose monitoring using implanted nanosensors, with key techniques being fluorescence resonance energy transfer (FRET) and fluorescence lifetime sensing, as well as new nano‐encapsulation technologies for sensors such as layer‐by‐layer (LBL) films. The latter might also achieve better insulin delivery in diabetes by both improved islet encapsulation and oral insulin formulations. An ‘artificial nanopancreas’ could be an alternative closed‐loop insulin delivery system. Other applications of nanomedicine include targeted molecular imaging in vivo (e.g. tissue complications) using quantum dots (QDs) or gold nanoparticles, and single‐molecule detection for the study of molecular diversity in diabetes pathology. Copyright

Coaxial nanosecond flashlamp

We have developed a coaxial thyratron‐gated flashlamp capable of being operated with high stability over a much wider range of conditions than previous flashlamps. Novel methods for continuously monitoring discharge stability are described. We present comprehensive data on the dependence of pulse width and intensity on operating parameters. Using the single photon detection technique an overall instrumental profile of 950 ps FWHM and 630 ps rise time has been obtained using hydrogen as a filler gas. An instrumental width of 1.2 ns FWHM can be routinely obtained in nitrogen even at a repetition rate of 100 kHz. Intensities of ≳109 photons per pulse in nitrogen and ≳108 in hydrogen can be obtained at the minimum pulse duration. The maximum intensity per pulse without added capacitance is ≳1010 photons. This performance is considerably better than that reported for conventional flashlamps.

Femtosecond two-photon excited fluorescence of melanin.

Abstract. Fluorescence of synthetic melanin in dimethyl sulfoxide has been excited by two‐photon absorption at 800 run, using 120 fs pulses with photon flux densities 1027 cm 2 s1. The shortest main component of the three‐exponential decay of fluorescence is 200 ± 2 ps. The overall spectral shape is red‐shifted with respect to the 400 nm excited fluorescence. Two‐photon excited melanin fluorescence also has been measured from excised samples of healthy human skin tissue. Because of the selectivity of melanin excitation via resonant two‐photon absorption, it is hypothesized that fluorescence excited in this way may yield information on malignant transformation.

In vivo glucose sensing for diabetes management: progress towards non-invasive monitoring

A device for continuous in vivo monitoring of glucose concentration in people with diabetes has been a clinical and research priority for many years but now has an urgency which is probably unquestioned in diabetes care. The purpose of this article is to explain recent advances in technology that are bringing glucose sensors closer to routine use and to highlight some of the remaining problems. Important new technologies include artificial receptors for glucose, tissue fluid sampling techniques, and new approaches to non-invasive sensing, such as fluorescence lifetime measurements.

Single-photon timing detectors for fluorescence lifetime spectroscopy

We review the principle of operation, performance and application of the different types of single-photon timing detectors presently employed in fluorescence lifetime determination using the time-correlated single-photon counting technique. The devices discussed include side-on and linear focused photomultiplier tubes, microchannel plate photomultipliers and avalanche photodiodes.

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.

Optothermal transient emission radiometry

Reports the development of a new technique-optothermal transient emission radiometry (OTTER)-for studying optical absorption, thermal diffusivities and associated properties in opaque materials. In essence, the technique makes use of the information content of the thermal emission transient observed by means of a wideband infrared detector, following pulsed optical excitation of the material. Some preliminary observations, including a study of several commercial pigments are presented.

Multiphoton excited fluorescence spectroscopy of biomolecular systems.

Recent work on the emerging application of multiphoton excitation to fluorescence studies of biomolecular dynamics and structure is reviewed. The fundamental principles and experimental techniques of multiphoton excitation are outlined, fluorescence lifetimes, anisotropy and spectra in membranes, proteins, hydrocarbons, skin, tissue and metabolites are featured, and future opportunities are highlighted.

Gold nanorods for fluorescence lifetime imaging in biology

Two-photon luminescence (TPL) from gold nanorods shows considerable potential in biological imaging. We study the imaging of gold nanorods in Madin-Darby canine kidney (MDCK) cells using fluorescence lifetime imaging microscopy (FLIM). FLIM provides images with better contrast and sensitivity than intensity imaging. The characteristic fluorescence lifetime of gold nanorods is found to be less than 100 ps, which can be used to distinguish gold nanorods from other fluorescent labels and endogenous fluorophores in lifetime imaging.