William R. Ware

Kinetics of diffusion‐controlled reactions: Transient effects in fluorescence quenching

A test of the continuum model for diffusion controlled reactions as applied to fluorescence quenching is presented and discussed. By proper selection of systems, it was possible to observe nonexponential decay which followed the decay law exp(−at − 2b√t) as predicted by the continuum model. Analysis of these decay curves gave the encounter radius and pair diffusion coefficient. Reasonable values were obtained for both. Steady−state fluorescence quenching measurements were also made and compared with fluorescence decay studies. Evidence was found for ground−state complex formation. When this was taken into account, good agreement between the continuum model and the experimental results was obtained.

A fallacy in the interpretation of fluorescence decay parameters

It is shown that single-photon-counting fluorescence decay data fit to a two-component decay law yielding satisfacory fit statistics and random residuals can conceal underlying sets of lifetimes with a variety of intensity distribution functions of remarkable width and diversity of shape. Only by collecting data to a large number of counts per channel (5 × 104 to 5 × 105 in the peak channel) with equipment free of common faults can one see even a hint of the presence of an underlying complex distribution of decay times. This conclusion has important and disturbing implications in studies where fluorescent molecules can emit under a variety of environmental conditions such as in proteins, on surfaces, in micelles and membranes, in cold beams clustered with other molecules, in polymers, etc.

Kinetics of partly diffusion controlled reactions. I. Transient and apparent transient effect in fluorescence quenching

Abstract Analysis of decay curves of electronically excited molecules A* versus time in presence of quencher B leads to the determination of kinetic data for the reaction (i.e. for diffusion limited reactions, the experimental collisional distance σ′ greater than the true collisional distance σ) and the sum D of diffusion coefficients of both reactants). Experimental fluorescence steady state measurements (Stern-Volmer representation) are inconsistent with calculated curves using classical Smoluchowskis model with σ′and D. The difference between this has been interpreted by complex formation in the ground state between A and B. But, this assumption is unnecessary if we take into account a “static” quenching arising from B molecules located at distances between σ and σ′ from A*. A theoretical model based upon this principle is described; good agreement between the model and experimental results was obtained.

Recovery of underlying distributions of lifetimes from fluorescence decay data

Abstract Fluorescence decay curves exhibiting good deconvolution fits to two or three exponentials can be interpreted in terms of two or three distinct lifetimes or as the result of complex underlying distributions which are not evident because of statistical noise. This work deals with the recovery of complex distributions of lifetimes from single photon decay data through the use of an exponential series probe function with fixed lifetimes and variable pre-exponential coefficients.

The Measurement of Short‐Lived Fluorescence Decay Using the Single Photon Counting Method

Refinements of the single photon technique are reported, which are concerned for the most part with the photon timing photomultiplier, its associated circuitry, and the signal processing equipment between this multiplier and the time to amplitude convertor. A photomultiplier voltage distribution circuit is described which permits one to optimize the performance of the photon counting photomultiplier either for sensitivity, time resolution, or both in order to assure the validity of the convolution integral as the representation of the instrument output. An investigation of the variation of the instrumental response with stop discriminator level is reported, and the origin and elimination of wavelength effects and photocathode area effects associated with the timing photomultiplier are also described.

Stroboscopic optical boxcar technique for the determination of fluorescence lifetimes

This work describes a stroboscopic optical boxcar technique for the determination of fluorescence lifetimes which achieves performance comparable to techniques such as time‐correlated single photon counting or phase modulation. The stroboscopic technique is based on the use of a traveling wave injected into a delay line connecting the dynodes of a photomultiplier tube. The transient potential difference created between two adjacent dynodes results simultaneously in significant amplification and the generation of a ‘‘gate’’ for the amplification process. Accurate control of the timing between the flashing of the gated lamp and the computer controlled delayable triggering of the photomultiplier tube pulser allows the gate to be placed at any time position within the range of the digital delay generator. The intensity of the fluorescence emission can thus be measured as a function of time relative to the excitation flash yielding data which is very similar to that from time‐correlated single photon counting....

Performance characteristics of a small side‐window photomultiplier in laser single‐photon fluorescence decay measurements

The performance characteristics of the Hamamatsu R928 side‐window photomultiplier tube have been evaluated in the picosecond time domain. This phototube is proposed as an attractive solution to the problem of high time resolution when a sync‐pumped dye laser is combined with the single‐photon time correlation method for determining fluorescence lifetimes and measuring time‐resolved emission spectra and polarization anisotropy. It combines excellent timing characteristics in the single‐photon mode and a small‐to‐negligible wavelength effect with high gain, low noise, and low cost. Twice the resolution previously reported has been obtained.

Fluorescence lifetimes of saturated hydrocarbons

Abstract Fluorescence lifetimes are reported for several linear and cyclic saturated hydrocarbons. Measurements were made with a new fluorescence lifetime apparatus designed for the vacuum ultraviolet. The results are in general in good agreement with those obtained by pulsed X-ray excitation and are shorter by about an order of magnitude than those determined by fluorescence quenching with the assumption of the Stokes-Einstein-Smoluchowski equation.

Biphasic photochemistry: time-resolved spectra of adsorbed hydrocarbons

Abstract Time-resolved fluorescence spectra were measured for pyrene and naphthalene adsorbed on a silica gel surface using the time-correlated photon-counting technique Excimer-Iike emission was observed within an excitation lamp duration of a few nanoseconds with both pyrene and naphthalene. It is suggested, from the excitation spectra, that ground state complexes of the adsorbates are responsible for the formation of the excimer-like emission.

Wavelength Effects in Fluorescence Quenching. Aromatic Hydrocarbons Quenched by Carbon Tetrachloride

The origin of wavelength effects in the fluorescence quenching of aromatic hydrocarbons by chlorinated alkanes has been investigated using fluorescence lifetime and fluorescence steady‐state techniques and absorption spectroscopy. Evidence was obtained for the presence of ground‐state complexes between the aromatic hydrocarbon and the chlorinated alkane in both polar and nonpolar solvents. The kinetics of the fluorescence quenching are consistent with the hypothesis that these ground‐state complexes are responsible for the variations of the quenching efficiency with wavelength.