Time-correlated Single Photon Counting Experiments Research Articles

Insight into Sodium Pump Regulation by FXYD Proteins

The sodium pump (Na+/K+-ATPase, NKA) is a membrane transport ATPase responsible for establishing the vital gradients of sodium and potassium across the plasma membrane of all mammalian cells. NKA activity is regulated by FXYD proteins, a family of transmembrane proteins that serve as auxiliary subunits of the sodium pump. in the heart, NKA activity is modulated by phospholemman (FXYD1), but the structural determinants of this regulatory mechanism are incompletely understood. We used time-resolved and steady-state FRET techniques to study the stoichiometry and affinity of fluorescently labeled NKA and FXYD1. Progressive acceptor photobleaching revealed multiple acceptor-labeled FXYD1 bound to donor-labeled NKA. Complementary global analysis of time-correlated single photon counting experiments revealed presence of medium- and high-FRET species within the population of NKA-FXYD1 complexes. To explore the quaternary structure of the regulatory complex, we obtained fluorescence lifetime data from hundreds of cells spanning a wide range of protein expression level. Over this range, the high FRET species increased with increasing protein concentration at the expense of the medium-FRET population. We attribute these subpopulations to NKA-NKA dimers decorated with either one FXYD1 (at low concentration) or two (at high concentration). Moreover, acceptor photobleaching experiments revealed FRET between donor- and acceptor-labeled NKA α subunits consistent with NKA oligomerization. in addition, we quantified FRET between NKA α and β essential subunits to monitor changes in the dynamic equilibrium between open (E2) and closed (E1) states of NKA. Treatment of NKA with the specific inhibitor ouabain induced a shift in the dynamic equilibrium of NKA toward a higher FRET state, suggesting that the α and β n-termini moved closer together. The experiments reveal new insight into the regulation of NKA by FXYD proteins and by NKA oligomerization.

DNA Hairpin Hybridization under Extreme Pressures: A Single-Molecule FRET Study.

Organisms have evolved to live in a variety of complex environments, which clearly has required cellular biology to accommodate to extreme conditions of hydraulic pressure and elevated temperature. In this work, we exploit single-molecule Forster resonance energy transfer (FRET) spectroscopy to probe structural changes in DNA hairpins as a function of pressure and temperature, which allows us to extract detailed thermodynamic information on changes in free energy (ΔG°), free volume (ΔV°), enthalpy (ΔH°), and entropy (ΔS°) associated with DNA loop formation and sequence-dependent stem hybridization. Specifically, time-correlated single-photon counting experiments on freely diffusing 40A DNA hairpin FRET constructs are performed in a 50 μm × 50 μm square quartz capillary cell pressurized from ambient pressure up to 3 kbar. By pressure-dependent van't Hoff analysis of the equilibrium constants, ΔV° for hybridization of the DNA hairpin can be determined as a function of stem length (nstem = 7-10) with single base-pair resolution, which further motivates a simple linear deconstruction into additive stem (ΔV°stem = ΔV°bp x nstem) and loop (ΔV°loop) contributions. We find that increasing pressure destabilizes the DNA hairpin stem region [ΔV°bp = +1.98(16) cm3/(mol bp)], with additional positive free volume changes [ΔV°loop = +7.0(14) cm3/mol] we ascribe to bending and base stacking disruption of the 40-dA loop. From a van't Hoff temperature-dependent analysis of the DNA 40A hairpin equilibria, the data support a similar additive loop/stem deconstruction of enthalpic (ΔH° = ΔH°loop + ΔH°stem) and entropic (ΔS° = ΔS°loop + ΔS°stem) contributions, which permits insightful comparison with predictions from nearest-neighbor thermodynamic models for DNA duplex formation. In particular, the stem thermodynamics is consistent with exothermically favored (ΔH°stem < 0) and entropically penalized (ΔS°stem < 0) hydrogen bonding but with additional enthalpic (ΔH°loop > 0) and entropic (ΔS°loop > 0) contributions due to loop bending effects consistent with distortion of dA base stacking in the 40-dA linker.

Fast fully-integrated front-end circuit to overcome pile-up limits in time-correlated single photon counting with single photon avalanche diodes.

Time-Correlated Single Photon Counting (TCSPC) is an essential tool in many scientific applications, where the recording of optical pulses with picosecond precision is required. Unfortunately, a key issue has to be faced: distortion phenomena can affect TCSPC experiments at high count rates. In order to avoid this problem, TCSPC experiments have been commonly carried out by limiting the maximum operating frequency of a measurement channel below 5% of the excitation frequency, leading to a long acquisition time. Recently, it has been demonstrated that matching the detector dead time to the excitation period allows to keep distortion around zero regardless of the rate of impinging photons. This solution paves the way to unprecedented measurement speed in TCSPC experiments. In this scenario, the front-end circuits that drive the detector play a crucial role in determining the performance of the system, both in terms of measurement speed and timing performance. Here we present two fully integrated front-end circuits for Single Photon Avalanche Diodes (SPADs): a fast Active Quenching Circuit (AQC) and a fully-differential current pick-up circuit. The AQC can apply very fast voltage variations, as short as 1.6ns, to reset external custom-technology SPAD detectors. A fast reset, indeed, is a key parameter to maximize the measurement speed. The current pick-up circuit is based on a fully differential structure which allows unprecedented rejection of disturbances that typically affect SPAD-based systems at the end of the dead time. The circuit permits to sense the current edge resulting from a photon detection with picosecond accuracy and precision even a few picoseconds after the end of the dead time imposed by the AQC. This is a crucial requirement when the system is operated at high rates. Both circuits have been deeply characterized, especially in terms of achievable measurement speed and timing performance.

Biomembrane Permeabilization: Statistics of Individual Leakage Events Harmonize the Interpretation of Vesicle Leakage.

The mode of action of membrane-active molecules, such as antimicrobial, anticancer, cell penetrating, and fusion peptides and their synthetic mimics, transfection agents, drug permeation enhancers, and biological signaling molecules (e.g., quorum sensing), involves either the general or local destabilization of the target membrane or the formation of defined, rather stable pores. Some effects aim at killing the cell, while others need to be limited in space and time to avoid serious damage. Biological tests reveal translocation of compounds and cell death but do not provide a detailed, mechanistic, and quantitative understanding of the modes of action and their molecular basis. Model membrane studies of membrane leakage have been used for decades to tackle this issue, but their interpretation in terms of biology has remained challenging and often quite limited. Here we compare two recent, powerful protocols to study model membrane leakage: the microscopic detection of dye influx into giant liposomes and time-correlated single photon counting experiments to characterize dye efflux from large unilamellar vesicles. A statistical treatment of both data sets does not only harmonize apparent discrepancies but also makes us aware of principal issues that have been confusing the interpretation of model membrane leakage data so far. Moreover, our study reveals a fundamental difference between nano- and microscale systems that needs to be taken into account when conclusions about microscale objects, such as cells, are drawn from nanoscale models.

Readout Architectures for High Efficiency in Time-Correlated Single Photon Counting Experiments—Analysis and Review

In recent years, time-correlated single photon counting (TCSPC) has become the technique of choice in many life science analyses, where fast and faint luminous signals are recorded with picosecond accuracy. Nevertheless, the maximum operating frequency of a single TCSPC acquisition channel limits the measurement speed, especially when scanning point systems are exploited. In order to increase the speed of TCSPC experiments, many multichannel systems based on single photon avalanche diode arrays have been proposed in the literature, which integrate thousands of pixels on the same chip. Unfortunately, the huge number of data generated by this kind of system can easily bring to the saturation of the transfer bandwidth to the external processing unit. For this reason, several different readout architectures have been proposed in the literature, attempting to exploit at best the limited bandwidth under TCSPC operating conditions. In this paper, some typical readout approaches, namely clock-driven and event-driven readouts, are discussed and compared, along with a recently-introduced router-based algorithm that is specifically designed to obtain maximum bandwidth exploitation under any condition. Quantitative comparisons are performed starting from imager response of the systems, which is the rate of recorded events in the case of uniform illumination of the detector array.

Photophysics of Pyrenyl-Functionalized Poly(isobutylene-alt-maleic anhydride) and Poly(isobutylene-alt-maleic N-alkylimide). Influence of Solvent, Degree of Substitution, and Temperature

A series of polymers derived from poly(isobutylene-alt-maleic anhydride) (PIMA) with ca. 1% to >90% of the anhydride units randomly substituted with 1-pyrenylmethylimido groups (Py-PIMA) has been synthesized and characterized. The remaining anhydride units in the Py-PIMA with 10% pyrenyl substitution have been converted to N-hexyl and N-decyl imides. The photophysical properties of these polymers, as obtained from steady-state fluorescence intensities, time-correlated single photon counting experiments, and time-resolved emission spectra, have provided insights into the dependence of the polymer conformations and their labilities on the degree of pyrenyl substitution, the nature of the appended alkyl chains, temperature, and solvent properties according to the Flory interaction coefficient and Hansen solubility parameters. None of the solvent characteristics, alone, can account for all aspects of the polymer behavior. The interactions of the medium with the polymer chains and the excited singlet states of...

Dispersed dynamics of solvation in G-quadruplex DNA: comparison of dynamic Stokes shifts of probes in parallel and antiparallel quadruplex structures

G-quadruplex DNA (GqDNA) structures play an important role in many specific cellular functions and are promising anti-tumor targets for small molecules (ligands). Here, we measured the dynamic Stokes shift of a ligand (Hoechst) bound to parallel c-Myc (mPu22) GqDNA over five decades of time from 100 fs to 10 ns, and compared it with the previously reported dynamics of DAPI bound to antiparallel human telomeric (hTelo22) GqDNA (Pal et al 2015 J. Phys. Chem. Lett. 6 1754). Stokes shift data from fluorescence up-conversion and time-correlated single photon counting experiments was combined to cover the broad dynamic range. The results show that the solvation dynamics of Hoechst in parallel mPu22 GqDNA follow a power law relaxation, added to fast 2 ps exponential relaxation, from 100 fs to 10 ns, with only a subtle difference of power law exponents in the two ligand-GqDNA systems (0.06 in Hoechst-mPu22 compared to 0.16 in DAPI-hTelo22). We measured steady-state fluorescence spectra and time-resolved anisotropy decays which confirm the tight binding of Hoechst to parallel mPu22 with a binding constant of ~1 × 105 M−1. The molecular docking of Hoechst in parallel GqDNA followed by a 50 ns molecular dynamics (MD) simulation on a Hoechst-GqDNA complex reveals that Hoechst binds to one of the outer G-tetrads by end-stacking near G13 and G4, which is different from the binding site of DAPI inside a groove of antiparallel hTelo22 GqDNA. Reconciling previous experimental and simulation results, we assign the 2 ps component to the hydration dynamics of only weakly perturbed water near mPu22 and the power law relaxation to the coupled motion of water and DNA (i.e. DNA backbone, unpaired bases and loops connecting G-tetrads) which come near the Hoechst inside parallel GqDNA.

Improving the counting efficiency in time-correlated single photon counting experiments by dead-time optimization.

Time-Correlated Single Photon Counting (TCSPC) has been long recognized as the most sensitive method for fluorescence lifetime measurements, but often requiring "long" data acquisition times. This drawback is related to the limited counting capability of the TCSPC technique, due to pile-up and counting loss effects. In recent years, multi-module TCSPC systems have been introduced to overcome this issue. Splitting the light into several detectors connected to independent TCSPC modules proportionally increases the counting capability. Of course, multi-module operation also increases the system cost and can cause space and power supply problems. In this paper, we propose an alternative approach based on a new detector and processing electronics designed to reduce the overall system dead time, thus enabling efficient photon collection at high excitation rate. We present a fast active quenching circuit for single-photon avalanche diodes which features a minimum dead time of 12.4 ns. We also introduce a new Time-to-Amplitude Converter (TAC) able to attain extra-short dead time thanks to the combination of a scalable array of monolithically integrated TACs and a sequential router. The fast TAC (F-TAC) makes it possible to operate the system towards the upper limit of detector count rate capability (∼80 Mcps) with reduced pile-up losses, addressing one of the historic criticisms of TCSPC. Preliminary measurements on the F-TAC are presented and discussed.

Nanoparticle Sizing and Potential Quality Control of Sols Using a Unique Fluorescence Anisotropy Probe and 3D Contour Anisotropy Mapping.

Spectroscopic properties of the particle sizing fluorophore Dipole Blue are reported. The probe is cationic in nature, highly water-soluble, and strongly adheres to anionic silica surfaces by electrostatic interactions, as is demonstrated here by Ludox SM 30. The probe has a distinct absorbance band centered at 320 nm, and the fluorescence emission band is Stokes-shifted 100 nm with a peak centered at 426 nm. From time-correlated single-photon counting experiments, the fluorescence lifetime was found to be adequately described by a three-exponential decay model with an intensity-averaged lifetime of 15.6 ns. Perrin graph analysis of steady-state anisotropy shows the presence of silica particles with a radius of (5.44 ± 0.16) nm, which, considering the distribution of particle sizes, is in reasonable agreement with 3.5 nm found from dynamic light scattering experiments.

Intramolecular cycloadditions of photogenerated azaxylylenes: an experimental and theoretical study.

The mechanism of intramolecular cycloadditions of azaxylylenes photogenerated via excited-state intramolecular proton transfer (ESIPT) in aromatic o-amido ketones and aldehydes bearing unsaturated functionalities was studied experimentally and computationally. In time-correlated single-photon counting experiments, no relation was found between lifetimes of singlet species and the nature of the amide pendant, either unsaturated furanpropanamide, capable of photocyclization, or the acetamide control. Steady-state emission for amido-tetralone derivatives showed comparable dual emission bands, but bromo substitution decreased the intensity of the ESIPT band. The most reactive derivatives of amidobenzaldehydes were virtually lacking the ESIPT band. The quantum yield of cycloaddition is decreased in the presence of triplet quenchers, O2 or trans-piperylene, and improved with heavy atom substitution in the aromatic ring, providing further evidence for the initial mechanistic hypothesis in which the fast singlet-state ESIPT is accompanied by the ISC in the tautomer (azaxylylene), which undergoes stepwise addition to the tethered unsaturated pendants.

Photoisomerization Kinetics of IR125 and HDITCP in Ionic Liquids with Different Cation Alkyl Chain Lengths

The photoisomerization kinetics of IR125 and HDITCP in ionic liquids with different cation alkyl chain lengths were investigated by measuring their fluorescence lifetimes and quantum yields using steady-state absorption and fluorescence spectroscopies, and time-correlated single-photon counting experiments. It was found that the photoisomerization rate constants for IR125 and HDITCP in all the selected ionic liquids were almost identical and did not change with increasing ionic liquid viscosity. A comparison of the photoisomerization rate constants of IR125 and HDITCP in isoviscous aqueous glycerol solutions with those in ionic liquids showed that the photoisomerization energy barriers of IR125 and HDITCP in ionic liquids were about 2 kJ∙mol-1 higher than those in the isoviscous aqueous glycerol solutions, indicating that specific interactions between IR125 or HDITCP and the ionic liquid restrain their respective photoisomerization processes in highly viscous ionic liquids.

Time-of-Flight Photon Spectroscopy: A Simple Scheme To Monitor Simultaneously Spectral and Temporal Fluctuations of Emission on Single Nanoparticles

Here we report on a novel scheme for spectral analysis that exploits the wavelength dependence of the time-of-flight of a photon in a dispersive medium. This versatile and cost-effective method, named time-of-flight photon spectroscopy (TOFPS), has the major advantage of being compatible with time-correlated single-photon counting experiments. Consequently, each photon acquired during an experiment is characterized by two parameters, its absolute time of arrival and its color, respectively. As a result, the spectral and temporal fluctuations of the emission of a single nano-object can be derived from a single measurement. As a proof of the concept, we demonstrate in the paper that the method can be used to perform Raman spectroscopy as well as fluorescence spectroscopy. We emphasize that TOFPS proves to be very efficient for improving signal-to-noise ratio in fluorescence correlation spectroscopy measurements by subsequent spectral filtering and to record luminescence spectra from single metallic particles. We demonstrate that the opportunity of simultaneously recording spectral and temporal fluctuations could be used to sort particles of different shapes inside a sample. TOFPS furthermore allows developing a new type of time interval distribution analysis which correlates the time interval between two photons and their corresponding color shift. It is applied to the analysis of the two-photon excited luminescence of a single gold nanorod. This method has a potential for a broad range of applications, among which time-resolved SERS spectroscopy and analysis of the dynamics of emission processes can be handled with new statistical approaches based on the correlation of spectral and temporal fluctuations.

Conformational dynamics of the tetracycline-binding aptamer

The conformational dynamics induced by ligand binding to the tetracycline-binding aptamer is monitored via stopped-flow fluorescence spectroscopy and time-correlated single photon counting experiments. The fluorescence of the ligand is sensitive to changes within the tertiary structure of the aptamer during and after the binding process. In addition to the wild-type aptamer, the mutants A9G, A13U and A50U are examined, where bases important for regulation are changed to inhibit the aptamer’s function. Our results suggest a very fast two-step-mechanism for the binding of the ligand to the aptamer that can be interpreted as a binding step followed by a reorganization of the aptamer to accommodate the ligand. Binding to the two direct contact points A13 and A50 was found to occur in the first binding step. The exchange of the structurally important base A9 for guanine induces an enormous deceleration of the overall binding process, which is mainly rooted in an enhancement of the back reaction of the first binding step by several orders of magnitude. This indicates a significant loss of tertiary structure of the aptamer in the absence of the base A9, and underlines the importance of pre-organization on the overall binding process of the tetracycline-binding aptamer.

Study of steady state and time resolved photoluminescence of thiol capped CdS nanocrystalline powders dispersed in N,N-dimethylformamide

The microwave (MW) assisted synthesis of thiol capped cadmium sulfide (CdS) nanocrystallites/quantum dots (QDs) was performed through the reaction of cadmium acetate with thiourea in N,N-dimethylformamide (DMF) by keeping the MW irradiation time fixed (40 s) in the presence of a thiol containing capping agent. Three capping agents, namely, benzyl mercaptan (BM), 1-butanethiol (BT) and 2-mercaptoethanol (ME) were used. The concentration of the precursors was varied to check the change in the average size of the thiol capped CdS nanocrystals formed. The nanocrystallites were characterized by usual procedure. The UV–vis absorption spectra and the photoluminescence (PL) spectra of the CdS nanocrystalline powders dispersed in DMF were studied. It was observed that with increase in concentration of the capping agent (BM), there is a shift in the nature of emission (PL) from trap associated PL to the band edge luminescence in the case of BM capped CdS nanocrystalline powders dispersed in DMF possibly due to better surface passivation. The relative PL quantum yield of the thiol capped CdS nanocrystalline powders dispersed in DMF was calculated under various experimental conditions. Time-correlated single-photon counting experiments were performed to study the time-resolved photoluminescence of the CdS nanocrystalline powders dispersed in DMF. The observed emission decay profiles have been simulated using the multiexponential model. The emission decay profiles for thiol capped CdS nanocrystalline powders dispersed in DMF depend on the nature of the capping agents (thiols) used to passivate the CdS nanocrystallites. The time resolved PL studies show that the average values of PL lifetime are related to the size and size distribution of the prepared thiol capped CdS nanocrystallites.

Determination of the Hydrogen-Bonding Induced Local Viscosity Enhancement in Room Temperature Ionic Liquids via Femtosecond Time-Resolved Depleted Spontaneous Emission

The fluorescence depletion dynamics of Rhodamine 700 (R-700) molecules in room temperature ionic liquids (RTILs) 1-ethyl-3-methylimidazolium tetrafluoroborate ([emim][BF(4)]) and 1-hydroxyethyl-3-methylimidazolium tetrafluoroborate ([HOemim][BF(4)]) were investigated to determine the local viscosity of the microenvironment surrounding the fluorescent molecules, which is induced by strong hydrogen bonding interaction between cationic and anionic components in RTILs. The solvation and rotation dynamics of R-700 molecules in RTILs show slower time constants relative to that in conventional protic solvents with the same bulk viscosity, indicating that the probe molecule is facing a more viscous microenvironment in RTILs than in conventional solvents because of the strong hydrogen bonding interaction between cationic and anionic components. In addition, this effect is more pronounced in hydroxyl-functionalized ionic liquid than in the regular RTIL due to the presence of a hydroxyl group as a strong hydrogen bonding donor. The hydrogen-bonding-induced local viscosity enhancement effect related to the heterogeneity character of RTILs is confirmed by the nonexponential rotational relaxation of R-700 determined by time-correlated single photon counting (TCSPC). The geometry of hydrogen bonding complexes with different components and sizes are further optimized by density functional theory methods to show the possible hydrogen-bond networks. A model of the hydrogen-bonding network in RTILs is further proposed to interpret the observed specific solvation and local viscosity enhancement effect in RTILs, where most of the fluoroprobes exist as the free nonbonding species in the RTIL solutions and are surrounded by the hydrogen-bonding network formed by the strong hydrogen-bonding between the cationic and anionic components in RTIL. The optimized geometry of hydrogen bonding complexes with different components and sizes by density functional theory methods confirms the local viscosity enhancement effect deduced from fluorescence depletion and TCSPC experiments. The calculated interaction energies reveal the existence of the stronger hydrogen bonding network in RTILs (especially in hydroxyl-functionalized ionic liquid) than that in conventional protic solvent, which leads to the enhancement effect of local microviscosity, and therefore leads to the slow solvation and rotation dynamics of probe molecules observed in RTILs.

Extended Förster theory for determining intraprotein distances : Part III. Partial donor–donor energy migration among reorienting fluorophores

An extended Förster theory (EFT) is derived and outlined for electronic energy migration between two fluorescent molecules which are chemically identical, but photophysically non-identical. These molecules exhibit identical absorption and fluorescence spectra, while their fluorescence lifetimes differ. The latter means that the excitation probability becomes irreversible. Unlike the case of equal lifetimes, which is often referred to as, donor-donor energy migration (DDEM), the observed fluorescence relaxation is then no longer invariant to the energy migration process. To distinguish, the present case is therefore referred to as partial donor-donor energy migration (PDDEM). The EFT of PPDEM is described by a stochastic master equation (SME), which has been derived from the stochastic Liouville equation (SLE) of motion. The SME accounts for the reorienting as well as the translational motions of the interacting chromophores. Synthetic fluorescence lifetime and depolarisation data that mimics time-correlated single photon counting experiments have been generated and re-analysed. The rates of reorientation, as well as the orientational configurations of the interacting D-groups were examined. Moreover the EFT of PPDEM overcomes the classical "kappa(2)-problem" and the frequently applied approximation of kappa(2) = 2/3 in the data analyses. An outline for the analyses of fluorescence lifetime and depolarisation data is also given, which might prove applicable to structural studies of D-labelled macromolecules, e.g. proteins. The EFT presented here brings the analyses of PDDEM data to the same level of molecular detail as that used in ESR- and NMR-spectroscopy.

Tunable Visible-Light Emission from CdS Nanocrystallites Prepared under Microwave Irradiation

The microwave (MW)-assisted reaction of cadmium acetate with thiourea in N,N-dimethylformamide (DMF) was controlled by controlling the MW irradiation time in the presence of 1 -thioglycerol as a capping agent. The peak position of the absorption band of the CdS nanocrystallites, dispersed in chloroform, shifts toward longer wavelength with increasing MW irradiation time, indicating growth of particle size under prolonged MW irradiation. However, the peak position of absorption band remains at the same wavelength, and only the intensity of the absorption band is increased when the MW irradiation of the colloidal solution of CdS nanocrystallites in DMF is periodically interrupted, keeping the solution at room temperature (27 °C) before each irradiation. This suggests that the particle growth occurs only during the continuous irradiation of MW and stops when the system is cooled down. From the spectral absorption edge, the diameter of CdS nanoparticles has been estimated. Photoluminescence of the CdS nanocrystallites, dispersed in chloroform, observed in the visible range shifts toward higher wavelength with increased duration of MW irradiation. When the MW irradiation was repeated for a fixed duration in colloidal solution of CdS nanocrystallites in DMF, enhancement in PL intensity was noticed without any change in the emission peak position. The relative PL quantum yield of the CdS nanocrystallites was estimated under various experimental conditions. Time-correlated single-photon counting experiments were performed to study the time-resolved photoluminescence of CdS nanocrystallites. The observed emission decay profiles have been simulated by using the multiexponential model.

Soluble polyimides containing trans-diaminotetraphenylporphyrin: Synthesis and photoinduced electron transfer

5,15-Bis(4-aminophenyl)-10,20-diphenylporphyrin (trans-DATPP) was synthesized via the condensation of meso-(4-nitrophenyl)dipyrromethane and benzaldehyde. The further reaction with zinc acetylacetonate hydrate afforded zinc 5,15-bis(4-aminophenyl)-10,20-diphenylporphyrin (trans-ZnDATPP). A series of soluble polyimides based on trans-DATPP or trans-ZnDATPP, 2,2′-bis(trifluoromethyl)-4,4′-diaminobiphenyl (PFMB) and 4,4′-hexafluoroisopropylidenediphthalic anhydride (6FDA) at various ratios was then prepared. Some physical properties of these polyimides were measured. It was found that every polyimide containing either trans-DATPP or trans-ZnDATPP had higher viscosity than the polyimide without porphyrin unit. Furthermore, the polyimides with trans-ZnDATPP showed lower viscosity than the ones without trans-ZnDATPP at approximately the same porphyrin content. Glass transition temperatures (Tgs) of polyimides containing trans-ZnDATPP were higher than polyimides containing trans-DATPP, and both were higher than polyimide without porphyrin. Steady state fluorescence spectroscopy of these polymers revealed that the quantum yield of polymers increased with higher content of free base porphyrin in the polymer chain. Time-correlated single photon counting experiments indicated these polyimides could be used in photonic applications.

Abstracting reliable parameters from time-correlated single photon counting experiments: I. A consideration of how many photons need to be collected and a simple pileup error correction for poly-exponential decay profiles without convolution of the lamp profile

An investigation of how many photons need to be collected, when using the time-correlated single photon counting method, in order to reproduce, within a reasonable accuracy, the generating function of a poly-exponential decay, indicates that this number is quite often considerably greater than that usually published. This poses a series of potential problems if considerably longer counting times are used. It is proposed that the solution is to collect data with substantial pileup. An analytical expression is given for correcting the errors due to pileup in poly-exponential decay profiles. The method, which requires only a knowledge of the STOP/START ratio and the experimental decay profiles, generates results basically identical to those obtained when pileup is intentionally limited, even for high pileup distortion, thus greatly reducing the time necessary to make measurements.

Utility and Considerations of Donor–Donor Energy Migration as a Fluorescence Method for Exploring Protein Structure-Function

This review aims at surveying the use of electronic energy transport between chemically identical fluorophores (i.e. donors) in studies of various protein systems. Applications of intra- and interprotein energy migration are presented that make use of polarised steady-state and time-resolved fluorescence spectroscopic techniques. The donor-donor energy migration (DDEM) and the partial donor-donor energy migration (PDDEM) models for calculating distances between donor groups are exposed together with the most recent development of an extended Forster theory (EFT). Synthetic fluorescence depolarisation data that mimic time-correlated single photon counting experiments were generated using the EFT, and then further re-analysed by the different models. The results obtained were compared with the known parameters used to generate EFT data. Aspects on how to adopt the EFT in the analyses of time-correlated single photon counting experiments are also presented, as well as future aspects on using energy migration for examining protein structure.