Time-resolved measurements are widely used in fluorescence spectroscopy, particularly for studies of biological macromolecules. This is because time-resolved data frequently contain more information than is available from the steady-state data. For instance, consider a protein which contains two tryptophan residues, each with a distinct lifetime. Because of spectral overlap of the absorption and emission, it is not usually possible to resolve the emission from the two residues. However, the time-resolved data may reveal two decay times, which can be used to resolve the emission spectra and relative intensities of the two tryptophan residues. Then one can question how each of the tryptophan residues is affected by the interactions of the protein with its substrate or other macromolecules. Is one of the tryptophan residues close to the binding site? Is a tryptophan residue in a distal domain affected by substrate binding to another domain? Such questions can be answered if one measures the decay times associated with each tryptophan residue.

https://link.springer.com/content/pdf/10.1007%2F978-1-4757-3061-6_4.pdf

Time-Domain Lifetime Measurements

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 differences in optical geometry can contribute to the lack of consistency between results obtained in different laboratories.

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

Formaldehyde, which is a generally occurring pollutant in the atmosphere and in building materials, can easily be determined by colorimetry or fluorimetry after derivatization to 3,5-diacetyl-1,4-dihydrolutidine (DDL) according to the Hantzsch reaction. Owing to the fact that the photophysical properties of DDL are to date widely unknown, the absorption and fluorescence behaviours as well as their changes under the influence of heat or irradiation have been investigated. From solvent-dependent measurements an increase in the dipole moment upon excitation is evident. In solvents of comparable polarity the spectra are more red shifted in protic media, which is probably due to hydrogen bonding. The fluorescence quantum yield was determined as φ f (20 °C) = 0.005 in aqueous solution and was found to be strongly dependent on temperature. The activation energy E A for non-radiative decay was determined according to an Arrhenius-type relationship. DDL decomposes on exposure to heat or light. The thermal degradation shows deviations from first-order kinetics and follows the time equation for an autocatalytic reaction. The quantum yield of the photochemical degradation was determined as Φ = (2–3) × 10 −3 .

Photophysical properties of 3,5-diacetyl-1,4-dihydrolutidine in solution: application to the analysis of formaldehyde

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.

Multiplexed time-correlated single-photon counting.

In acetonitrile, the photoreactions of cis-anethole, cA, or trans-anethole, tA, with fumarodinitrile, FN, lead to isomerization of both substrate and quencher and to mixed [2 + 2] cycloaddition By

Photoinduced Electron Transfer Reactions of Aryl Olefins. 2.† Cis−Trans Isomerization and Cycloadduct Formation in Anethole−Fumaronitrile Systems in Polar Solvents

T. Salthammer

Papers

Time-correlated single-photon counting with alternate recording of excitation and emission