Time-correlated Single Photon Counting Data Research Articles

Blind instrument response function identification from fluorescence decays

Time-resolved fluorescence spectroscopy plays a crucial role when studying dynamic properties of complex photochemical systems. Nevertheless, the analysis of measured time decays and the extraction of exponential lifetimes often requires either the experimental assessment or the modeling of the instrument response function (IRF). However, the intrinsic nature of the IRF in the measurement process, which may vary across measurements due to chemical and instrumental factors, jeopardizes the results obtained by reconvolution approaches. In this paper, we introduce a novel methodology, called blind instrument response function identification (BIRFI), which enables the direct estimation of the IRF from the collected data. It capitalizes on the properties of single exponential signals to transform a deconvolution problem into a well-posed system identification problem. To delve into the specifics, we provide a step-by-step description of the BIRFI method and a protocol for its application to fluorescence decays. The performance of BIRFI is evaluated using simulated and time-correlated single-photon counting data. Our results demonstrate that the BIRFI methodology allows an accurate recovery of the IRF, yielding comparable or even superior results compared with those obtained with experimental IRFs when they are used for reconvolution by parametric model fitting.

Fluorescence Correlation Spectroscopy Combined with Multiphoton Laser Scanning Microscopy—A Practical Guideline

Multiphoton laser scanning microscopy (MPM) has opened up an optical window into biological tissues; however, imaging is primarily qualitative. Cell morphology and tissue architectures can be clearly visualized but quantitative analysis of actual concentration and fluorophore distribution is indecisive. Fluorescence correlation spectroscopy (FCS) is a highly sensitive photophysical methodology employed to study molecular parameters such as diffusion characteristics on the single molecule level. In combination with laser scanning microscopy, and MPM in particular, FCS has been referred to as a standard and highly useful tool in biomedical research to study diffusion and molecular interaction with subcellular precision. Despite several proof-of-concept reports on the topic, the implementation of MPM-FCS is far from straightforward. This practical guideline aims to clarify the conceptual principles and define experimental operating conditions when implementing MPM-FCS. Validation experiments in Rhodamine solutions were performed on an experimental MPM-FCS platform investigating the effects of objective lens, fluorophore concentration and laser power. An approach based on analysis of time-correlated single photon counting data is presented. It is shown that the requirement of high numerical aperture (NA) objective lenses is a primary limitation that restricts field of view, working distance and concentration range. Within these restrictions the data follows the predicted theory of Poisson distribution. The observed dependence on laser power is understood in the context of perturbation on the effective focal volume. In addition, a novel interpretation of the effect on measured diffusion time is presented. Overall, the challenges and limitations observed in this study reduce the versatility of MPM-FCS targeting biomedical research in complex and deep tissue—being the general strength of MPM in general. However, based on the systematic investigations and fundamental insights this report can serve as a practical guide and inspire future research, potentially overcoming the technical limitations and ultimately allowing MPM-FCS to become a highly useful tool in biomedical research.

High-accuracy and video-rate lifetime extraction from time correlated single photon counting data on a graphical processing unit

Graphical Processing Units (GPUs) are a powerful alternative to central processing units, especially for data-parallel, video-rate processing of large data volumes. In the complex scenario of high-performance, multichannel Time Correlated Single Photon Counting (TCSPC), a huge amount of data is potentially generated by the acquisition system. Exploiting a dedicated, external, programmable elaboration unit enables a high degree of flexibility to perform different types of analysis. In this paper, we present a GPU-based application that leverages the common unified device architecture application programming interface for video-rate and accurate lifetime extraction from TCSPC data acquired at a rate of up to 10 Gbit/s.

ReadPTU: a python library to analyse time tagged time resolved data

readPTU is a python package designed to analyze time-correlated single-photon counting data. The use of the library promotes the storage of the complete time arrival information of the photons and full flexibility in post-processing data for analysis. The library supports the computation of time resolved signal with external triggers and second order autocorrelation function analysis can be performed using multiple algorithms that provide the user with different trade-offs with regards to speed and accuracy. Additionally, a thresholding algorithm to perform time post-selection is also available. The library has been designed with performance and extensibility in mind to allow future users to implement support for additional file extensions and algorithms without having to deal with low level details. We demonstrate the performance of readPTU by analyzing the second-order autocorrelation function of the resonance fluorescence from a single quantum dot in a two-dimensional semiconductor.

Embedded Fluorescence Lifetime Determination for High-Throughput, Low-Photon-Number Applications

Time-resolved fluorescence (TRF) analysis is considered to be among the primary research tools in biochemistry and biophysics. One application of this method is the investigation of biomolecular interactions with promising applications for biosensing. For the latter context, time-correlated single photon counting (TCSPC) is the most sensitive, hence preferred implementation of TRF. However, high throughput applications are presently limited by the maximum achievable photon acquisition rate, and even more by the data processing rate. The latter rate is actually limited by the computational complexity to estimate accurately the fluorescence lifetime from TCSPC data. Here we propose a solution that would enable the implementation of TRF detection for fluorescence-activated droplet sorting (FADS), a particularly high throughput, microfluidic-based technology that can be used for drug discovery and thus help finding new cures for diseases. Most fluorescence lifetime algorithms require a large number of detected photons for an accurate lifetime computation. This paper presents an implementation based on a maximum likelihood estimator (MLE), enabling high precision estimation with a limited number of detected photons, significantly reducing the total measurement time. This speedup rapidly increases the input data rate. As a result, off-the-shelf embedded products cannot handle the data rates produced by current TCSPC units that are used to measure the fluorescence. Therefore, a configurable real-time capable hardware architecture is implemented on a field-programmable gate array (FPGA) that can handle the data rates of future TCSPC units, rendering high throughput droplet sorting with microfluidics possible. The presented hardware architecture is validated with experimental input data and produces high precision results.

Acceptor number-dependent ultrafast photo-physical properties of push-pull chromophores using time-resolved methods

Three push-pull chromophores comprising a triphenylamine (TPA) as electron-donating moiety and functionalized β-diketones as electron acceptor units are studied by various spectroscopic techniques. The time-correlated single-photon counting data shows that increasing the number of electron acceptor units accelerates photoluminescence relaxation rate of compounds. Transient spectra data shows that intramolecular charge transfer (ICT) takes place from TPA units to β-diketones units after photo-excitation. Increasing the number of electron acceptor units would prolong the generation process of ICT state, and accelerate the excited molecule reorganization process and the relaxation process of ICT state.

Excitation transfer and trapping kinetics in plant photosystem I probed by two-dimensional electronic spectroscopy.

Photosystem I is a robust and highly efficient biological solar engine. Its capacity to utilize virtually every absorbed photon's energy in a photochemical reaction generates great interest in the kinetics and mechanisms of excitation energy transfer and charge separation. In this work, we have employed room-temperature coherent two-dimensional electronic spectroscopy and time-resolved fluorescence spectroscopy to follow exciton equilibration and excitation trapping in intact Photosystem I complexes as well as core complexes isolated from Pisum sativum. We performed two-dimensional electronic spectroscopy measurements with low excitation pulse energies to record excited-state kinetics free from singlet-singlet annihilation. Global lifetime analysis resolved energy transfer and trapping lifetimes closely matches the time-correlated single-photon counting data. Exciton energy equilibration in the core antenna occurred on a timescale of 0.5ps. We further observed spectral equilibration component in the core complex with a 3-4ps lifetime between the bulk Chl states and a state absorbing at 700nm. Trapping in the core complex occurred with a 20ps lifetime, which in the supercomplex split into two lifetimes, 16ps and 67-75ps. The experimental data could be modelled with two alternative models resulting in equally good fits-a transfer-to-trap-limited model and a trap-limited model. However, the former model is only possible if the 3-4ps component is ascribed to equilibration with a "red" core antenna pool absorbing at 700nm. Conversely, if these low-energy states are identified with the P700 reaction centre, the transfer-to-trap-model is ruled out in favour of a trap-limited model.

An insight to the binding of ellagic acid with human serum albumin using spectroscopic and isothermal calorimetry studies

Ellagic acid (EA), a natural polyphenol evidence several pharmacological benefits. The binding profile of EA with human serum albumin (HSA) has been explored and investigated by Isothermal titration calorimetry (ITC), circular dichroism (CD) spectroscopy, time-correlated single-photon counting (TCSPC), absorbance spectroscopy, steady-state fluorescence spectroscopy, and modelling studies. The ITC data analysis revealed the binding Constant (Ka), ΔH, ΔS and ΔG values to be 15.5×104M−1, −116.2±18.1Kcalmol−1, −366calmol−1K−1 and −7.13Kcal mol−1 respectively with a unique binding site at HSA. EA effectively quenched the intrinsic fluorescence of HSA by static quenching, whereas TCSPC data also revealed association of dynamic quenching also. Thermodynamic analysis confirmed that hydrophobic and mainly hydrogen bonding interaction played important role in stabilizing the HSA-EA complex. It further dictates the binding reaction to be enthalpy driven. The secondary structure of HSA was altered upon binding with EA. CD spectroscopic data indicated the fraction of alpha helicity to be decreased from 52% to 40% upon binding to EA. This study will provide an insight on evaluation of this bioactive interaction during transport and releasing efficiency at the target site in human physiological system since HSA is the most important carrier protein in blood serum.

Temporal binning of time-correlated single photon counting data improves exponential decay fits and imaging speed.

Time-correlated single photon counting (TCSPC) enables acquisition of fluorescence lifetime decays with high temporal resolution within the fluorescence decay. However, many thousands of photons per pixel are required for accurate lifetime decay curve representation, instrument response deconvolution, and lifetime estimation, particularly for two-component lifetimes. TCSPC imaging speed is inherently limited due to the single photon per laser pulse nature and low fluorescence event efficiencies (<10%) required to reduce bias towards short lifetimes. Here, simulated fluorescence lifetime decays are analyzed by SPCImage and SLIM Curve software to determine the limiting lifetime parameters and photon requirements of fluorescence lifetime decays that can be accurately fit. Data analysis techniques to improve fitting accuracy for low photon count data were evaluated. Temporal binning of the decays from 256 time bins to 42 time bins significantly (p<0.0001) improved fit accuracy in SPCImage and enabled accurate fits with low photon counts (as low as 700 photons/decay), a 6-fold reduction in required photons and therefore improvement in imaging speed. Additionally, reducing the number of free parameters in the fitting algorithm by fixing the lifetimes to known values significantly reduced the lifetime component error from 27.3% to 3.2% in SPCImage (p<0.0001) and from 50.6% to 4.2% in SLIM Curve (p<0.0001). Analysis of nicotinamide adenine dinucleotide-lactate dehydrogenase (NADH-LDH) solutions confirmed temporal binning of TCSPC data and a reduced number of free parameters improves exponential decay fit accuracy in SPCImage. Altogether, temporal binning (in SPCImage) and reduced free parameters are data analysis techniques that enable accurate lifetime estimation from low photon count data and enable TCSPC imaging speeds up to 6x and 300x faster, respectively, than traditional TCSPC analysis.

Phosphorylation Modulates Conformational Bias of a Disordered Peptide

Disordered proteins, proteins lacking a stable three-dimensional structure, are often enriched in a broadly conserved sequence composition and patterning that imbues them with residual conformational bias toward the polyproline-II (PII) conformation. Since post-translational modification often effects biological function, we ask how phosphorylation impacts PII bias and overall geometry of a short, phosphorylatable peptide. We developed a method to use time-correlated single-photon counting data to measure Forster Resonance Energy Transfer (FRET), then subsequently calculate polymer properties of the peptide. We explore the impact of phosphorylation on effective persistence length and end-to-end distance, and compare the results of our experiments to behaviors modeled by a hard-sphere collision simulation.

Spectral decomposition of NAD(P)H fluorescence components recorded by multi-wavelength fluorescence lifetime spectroscopy in living cardiac cells

We report a novel analytical approach to identify individual components of a cell’s endogenous fluorescence, recorded by spectrally-resolved time-correlated single photon counting (TCSPC). Time-resolved area-normalized emission spectroscopy (TRANES) and principal component analysis (PCA) were applied to estimate the number of spectral components after metabolic modulation of cardiac cells following excitation with a 375 nm picosecond laser. Linear unmixing of TCSPC data spectrally decomposed individual components in living cells, while using characteristics of endogenously fluorescing molecules in solvents as a reference spectral database. Our data demonstrate the presence of three individual components, corresponding to the nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) in organic and inorganic solvents and to the residual flavoprotein fluorescence. The presented analytical approach offers a new alternative for the spectral separation of multi-wavelength fluorescence lifetime spectroscopy data to the conventional analysis, and opens a new possibility for the use of pattern recognition for fast resolution of components in 2D fluorescence lifetime microscopy images.

Dielectric Relaxation and Solvation Dynamics in a Prototypical Ionic Liquid + Dipolar Protic Liquid Mixture: 1-Butyl-3-Methylimidazolium Tetrafluoroborate + Water

Dielectric and solvation data on mixtures of 1-butyl-3-methylimidazilium tetrafluoroborate ([Im41][BF4]) + water are reported and used to examine the utility of dielectric solvation models. Dielectric permittivity and loss spectra (25 °C) were recorded over the frequency range 200 MHz to 89 GHz at 17 compositions and fit to a 4-Debye form. Dynamic Stokes shift measurements on the solute coumarin 153 (C153), made by combining fluorescence upconversion (80 fs resolution) and time-correlated single photon counting data (20 ns range), were used to determine the solvation response at 7 compositions (20.5 °C). All properties measured here were found to depend upon mixture composition in a simple continuous manner, especially when viewed in terms of volume fraction. Solvation response functions predicted by a simple dielectric continuum model are similar to but ∼7-fold faster than the spectral response functions measured with C153. The solvation data are in better agreement with the recently published predictions of a semimolecular model of Biswas and co-workers [J. Phys. Chem. B 2011, 115, 4011], but these latter predictions are systematically slow by a factor of ∼3.

Light soaking effects on charge recombination and device performance in dye sensitized solar cells based on indoline–cyclopentadithiophene chromophores

The synthesis, characterization, electrochemical and photophysical properties of a novel D–π–A indoline organic dye, VCL01, are described. Its performance characteristics in dye sensitized solar cell (DSC) devices under standard AM 1.5G illumination are also investigated. VCL01 incorporates a cyclopentadithiophene unit as the π-bridge between the indoline donor and cyanoacetic acceptor units. In comparison with the reference dye LS-1 containing only one thiophene unit in the π-bridge, VCL01 shows a 40 nm red shift in adsorption, an increase in molar absorptivity and a 0.17 V lower oxidation potential, all consistent with the more conjugated nature of this sensitizer. The efficiencies of VCL01 and LS-1 DSC devices were 4.81% and 6.23%, respectively, which upon >100 min continuous light soaking under AM 1.5G illumination rose to 7.21% and 6.95%, representing an unprecedented 50% increase in efficiency for the VCL01 device. This increase is overwhelmingly due to an increase in photocurrent but, remarkably, Voc also increases by 50 mV upon illumination reflected in transient photovoltage data which indicate that electron lifetime increases considerably also. Time-correlated single photon counting data indicate that the light soaking effect can be partly attributed to improved TiO2/dye interaction leading to enhanced electron injection.

Time variation of fluorescence lifetime in enhanced cyan fluorescence protein

The lifetime variations of enhanced cyan fluorescence protein (ECFP) in relatively short integration time bins were studied via time-correlated single photon counting (TCSPC) measurement. We observed that minimum photon counts are necessary for the lifetime estimation to achieve a certain range of variance. The conditions to decrease the variance of lifetime were investigated and the channel width of the measurement of TCSPC data was found to be another important factor for the variance of lifetime. Though the lifetime of ECFP is best fit by a double exponential, a mono exponential fit for the same integration time is more stable. The results may be useful in the analysis of photophysical dynamics for ensemble molecules in short measurement time windows.

Effect of Hydrogen Bonding on the Rotational and Translational Dynamics of a Headgroup-Bound Chromophore in Bilayer Lipid Membranes

We have studied the interactions of the chromophore 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-7-nitro-2-1,3-benzoxadiazol-4-yl (18:1 NBD-PE) imbedded in the headgroup region of bilayer lipid membranes consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DOPG). We have examined the molecular and mesoscale dynamics of the chromophore using time-correlated single photon counting (TCSPC) to measure rotational diffusion dynamics in lipid vesicles and fluorescence recovery after pattern photobleaching (FRAPP) to determine translational diffusion coefficients and mobile fractions in supported lipid bilayers. TCSPC data reveal that chromophore rotational diffusion rates in DOPG vesicles are statistically the same as in DOPC and mixed DOPC/DOPG vesicles, suggesting that the NBD-PE chromophore does not interact strongly with the headgroup region of these bilayers; however, FRAPP experiments show that lateral diffusion is statistically lower in mixed DOPC/DOPG-supported bilayers than in DOPC-supported bilayers. These results suggest that bilayers containing DOPG likely undergo interlipid headgroup hydrogen bonding interactions that suppress translational diffusion.

Global analysis of time correlated single photon counting FRET-FLIM data

Fluorescence lifetime imaging microscopy (FLIM) can be used to quantify molecular reactions in cells by detecting fluorescence resonance energy transfer (FRET). Confocal FLIM systems based on time correlated single photon counting (TCSPC) methods provide high spatial resolution and high sensitivity, but suffer from poor signal to noise ratios (SNR) that complicate quantitative analysis. We extend a global analysis method, originally developed for single frequency domain FLIM data, with a new filtering method optimized for FRET-FLIM data and apply it to TCSPC data. With this approach, the fluorescent lifetimes and relative concentrations of free and interacting molecules can be reliably estimated, even if the SNR is low. The required calibration values of the impulse response function are directly estimated from the data, eliminating the need for reference samples. The proposed method is efficient and robust, and can be routinely applied to analyze FRET-FLIM data acquired in intact cells.

Global fitting without a global model: Regularization based on the continuity of the evolution of parameter distributions

We introduce a new approach to global data fitting based on a regularization condition that invokes continuity in the global data coordinate. Stabilization of the data fitting procedure comes from probabilistic constraint of the global solution to physically reasonable behavior rather than to specific models of the system behavior. This method is applicable to the fitting of many types of spectroscopic data including dynamic light scattering, time-correlated single-photon counting (TCSPC), and circular dichroism. We compare our method to traditional approaches to fitting an inverse Laplace transform by examining the evolution of multiple lifetime components in synthetic TCSPC data. The global regularizer recovers features in the data that are not apparent from traditional fitting. We show how our approach allows one to start from an essentially model-free fit and progress to a specific model by moving from probabilistic to deterministic constraints in both Laplace transformed and nontransformed coordinates.

A two-dimensional view of the folding energy landscape of cytochrome c

Time-correlated single photon counting (TCSPC) was combined with fluorescence correlation spectroscopy (FCS) to study the transition between acid-denatured states and the native structure of cytochrome c (Cyt c) from Saccharomyces cerevisiae. The use of these techniques in concert proved to be more powerful than either alone, yielding a two-dimensional picture of the folding energy landscape of Cyt c. TCSPC measured the distribution of distances between the heme of the protein and a covalently attached dye molecule at residue C102 (one folding reaction coordinate), whereas FCS measured the hydrodynamic radius (a second folding reaction coordinate) of the protein over a range of pH values. These two independent measurements provide complimentary information regarding protein conformation. We see evidence for a well defined folding intermediate in the acid renaturation folding pathway of this protein reflected in the distribution of lifetimes needed to fit the TCSPC data. Moreover, FCS studies revealed this intermediate state to be in dynamic equilibrium with unfolded structures, with conformational fluctuations into and out of this intermediate state occurring on an approximately 30-micros time scale.

Photophysics of PTCDA and Me-PTCDI Thin Films: Effects of Growth Temperature

The effect of deposition temperature on the photophysical properties of 3,4,9,10-perylenetetracarboxylic dianhydride (PTCDA) and N,N'-dimethylperylene-3,4,9,10-bis(dicarboximide) (Me-PTCDI) films is investigated with steady-state and time-resolved spectroscopy. Atomic force microscopy (AFM) images of the film surfaces show an increase in the dimensions of crystallites with substrate temperature, culminating in the formation of elongated crystallites on substrates held close to the sublimation temperature. In contrast, despite an improvement in the crystal quality, X-ray diffraction (XRD) studies indicate that the substrate temperature has a negligible effect on the molecular orientation; the PTCDA and Me-PTCDI molecules align parallel and tilted to the substrate surface, respectively. Both materials exhibit characteristic absorption, due to mixing between charge-transfer and Frenkel species, and broad structureless photoluminescence. Growth at elevated temperatures gives rise to increased low-energy absorption, attributed to the formation of charge-transfer species, and enhanced blue-shifted emission, although the effects are less pronounced for Me-PTCDI. Time-correlated single-photon counting data indicate that the enhancement coincides with a lengthening of the fluorescence decays, over the whole emission spectrum.

Enhancing the Sensitivity of Fluorescence Correlation Spectroscopy by Using Time-Correlated Single Photon Counting

Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) are methods that extract information about a sample from the influence of thermodynamic equilibrium fluctuations on the fluorescence intensity. This method allows dynamic information to be obtained from steady state equilibrium measurements and its popularity has dramatically increased in the last 10 years due to the development of high sensitivity detectors and its combination with confocal microscopy. Using time-correlated single-photon counting (TCSPC) detection and pulsed excitation, information over the duration of the excited state can be extracted and incorporated in the analysis. In this short review, we discuss new methodologies that have recently emerged which incorporated fluorescence lifetime information or TCSPC data in the FCS and FCCS analysis. Time-gated FCS discriminates between which photons are to be incorporated in the analysis dependent upon their arrival time after excitation. This allows for accurate FCS measurements in the presence of fluorescent background, determination of sample homogeneity, and the ability to distinguish between static and dynamic heterogeneities. A similar method, time-resolved FCS can be used to resolve the individual correlation functions from multiple fluorophores through the different fluorescence lifetimes. Pulsed interleaved excitation (PIE) encodes the excitation source into the TCSPC data. PIE can be used to perform dual-channel FCCS with a single detector and allows elimination of spectral cross-talk with dual-channel detection. For samples that undergo fluorescence resonance energy transfer (FRET), quantitative FCCS measurements can be performed in spite of the FRET and the static FRET efficiency can be determined.