Optimized gating scheme for rapid lifetime determinations of single-exponential luminescence lifetimes

The rapid lifetime method (RLD) for determining excited-state lifetimes uses the ratio of the areas under two regions of the decay. To get good precision with the standard method, prior knowledge of the lifetime is essential to selecting the integration regions. As will be shown, the usual method of selecting integration regions is far from optimal. An optimal gating scheme that is more precise and much more forgiving in the selection of integration region than any of the prior methods will be shown. Monte Carlo simulations were performed to determine the optimal gating. Experimental data was used to confirm the capabilities of the optimized RLD. The speed of the optimal RLD is similar to the standard RLD but without the necessity of matching the integration interval to the lifetime for precise results.     

Luminescence lifetime standards for the nanosecond to microsecond range and oxygen quenching of ruthenium(II) complexes

A rapid and reproducible method for determining the temperature dependence of luminescence lifetimes has been developed. With the use of this method, a set of standards for the excited-state lifetime oxygen quenching of several ruthenium(II) transition metal complexes was established. With the use of three solvents of different viscosities and two metal complexes with widely different lifetimes, an overlapping range of ca. 100 ns to 6 micros was obtained. The decays are pure single exponentials, which means that they can be used reliably with both phase and pulsed lifetime instruments. For a pure single-exponential decay, a properly operating phase shift instrument will give the same lifetime as a time domain instrument. With the use of a thermal deactivation model and a three-parameter temperature-dependent oxygen quenching constant, the lifetime temperature-dependent data was well fit by a simple six-parameter equation that covers the temperature range of 10-50 degrees C and oxygen pressures from 0 to 1 atm of oxygen with excellent precision (ca. <1%). This permits both laboratory and field calibration of instruments.     

Dynamic windowing algorithm for the fast and accurate determination of luminescence lifetimes

An algorithm for the accurate calculation of luminescence lifetimes in near-real-time is described. The dynamic rapid lifetime determination (DRLD) method uses a window-summing technique and dynamically selects the appropriate window width for each lifetime decay such that a large range of lifetimes can be accurately calculated. The selection of window width is based on an optimal range of window-sum ratios. The algorithm was compared to alternative approaches for rapid lifetime determination as well as nonlinear least-squares (NLLS) fitting in both simulated and real experimental conditions. A palladium porphyrin was used as a model luminophore to quantitatively evaluate the algorithm in a dynamic situation, where oxygen concentration was modulated to induce a change in lifetime. Unlike other window-summing techniques, the new algorithm calculates lifetimes that are not significantly different than the slower, traditional NLLS. In addition, the computation time required to calculate the lifetime is 4 orders of magnitude less than NLLS and 2 orders less than other iterative methods. This advance will improve the accuracy of real-time measurements that must be made on samples that are expected to exhibit widely varying lifetimes, such as sensors and biosensors.     

Comparison of methods for rapid evaluation of lifetimes of exponential decays

For evaluating exponential luminescence decays, there are a variety of computational rapid integral methods based on the areas of the decay under different binned intervals. Using both Monte Carlo methods and experimental photon counting data, we compare the standard rapid lifetime determination method (SRLD), optimized rapid lifetime determination methods (ORLD), maximum likelihood estimator method (MLE), and the phase plane method (PPM). The different techniques are compared with respect to precision, accuracy, sensitivity to binning range, and the effect of baseline interference. The MLE provides the best overall precision, but requires 10 bins and is sensitive to very small uncorrected baselines. The ORLD provides nearly as good precision using only two bins and is much more immune to uncompensated baselines. The PPM requires more bins than the MLE and has systematic errors, but is largely resistant to baseline issues. Therefore, depending on the data acquisition method and the number of bins that can be readily employed, the ORLD and MLE are the preferred methods for reasonable signal-to-noise ratios.     

Effects of inaccurate reference lifetimes on interpreting frequency-domain fluorescence data

The effects of assigning inaccurate reference lifetimes in lifetime determinations are predicted theoretically by using standard equations. This theory leads to a method to remove reference error effects using common least-squares software. This method cannot, however, be used to deconvolute data collected with isochronal references. Uncorrected data can always be exactly solved with models containing one more degree of freedom than the true model. Monoexponential decays are fit by double-exponential decays or excited-state processes. Unimodal distributed decays often appear as discrete, double-exponential decays.     

Fluorescence lifetime standards for time and frequency domain fluorescence spectroscopy

A series of fluorophores with single-exponential fluorescence decays in liquid solution at 20 degrees C were measured independently by nine laboratories using single-photon timing and multifrequency phase and modulation fluorometry instruments with lasers as excitation source. The dyes that can serve as fluorescence lifetime standards for time-domain and frequency-domain measurements are all commercially available, are photostable under the conditions of the measurements, and are soluble in solvents of spectroscopic quality (methanol, cyclohexane, water). These lifetime standards are anthracene, 9-cyanoanthracene, 9,10-diphenylanthracene, N-methylcarbazole, coumarin 153, erythrosin B, N-acetyl-l-tryptophanamide, 1,4-bis(5-phenyloxazol-2-yl)benzene, 2,5-diphenyloxazole, rhodamine B, rubrene, N-(3-sulfopropyl)acridinium, and 1,4-diphenylbenzene. At 20 degrees C, the fluorescence lifetimes vary from 89 ps to 31.2 ns, depending on fluorescent dye and solvent, which is a useful range for modern pico- and nanosecond time-domain or mega- to gigahertz frequency-domain instrumentation. The decay times are independent of the excitation and emission wavelengths. Dependent on the structure of the dye and the solvent, the excitation wavelengths used range from 284 to 575 nm, the emission from 330 to 630 nm. These lifetime standards may be used to either calibrate or test the resolution of time- and frequency-domain instrumentation or as reference compounds to eliminate the color effect in photomultiplier tubes. Statistical analyses by means of two-sample charts indicate that there is no laboratory bias in the lifetime determinations. Moreover, statistical tests show that there is an excellent correlation between the lifetimes estimated by the time-domain and frequency-domain fluorometries. Comprehensive tables compiling the results for 20 (fluorescence lifetime standard/solvent) combinations are given.     

Direct observation of molecular recognition mediated by triple hydrogen bonds at a water/oil interface: time-resolved total internal reflection fluorometry study

Molecular recognition mediated by hydrogen-bonding interactions at a water/CCl(4) interface was investigated directly by means of time-resolved total internal reflection (TIR) fluorescence spectroscopy. The TIR fluorescence decay profile of riboflavin (RF) in the absence of a guest in the CCl(4) phase was fitted satisfactorily by a single-exponential function. In the presence of N,N-dioctadecyl-[1,3,5]triazine-2,4,6-triamine (DTT) as a guest in the CCl(4) phase, on the other hand, the fluorescence decay profiles were best fitted by double-exponential functions with the relevant amplitude (A(i)) being varied with the concentration of DTT. Furthermore, the rotational reorientation time of RF at the interface determined by fluorescence dynamic anisotropy was 210 ps in the absence of DTT, while fast (160-220 ps) and slow (670-750 ps) rotational reorientation times were observed in the presence of DTT. This slow rotational reorientation time was shown to ascribe to that of the RF-DDT complex formed at the water/CCl(4) interface. These results indicate that molecular recognition mediated by complementary hydrogen bonding takes place effectively at the water/CCl(4) interface, which was observed directly by both fluorescence dynamics and fluorescence dynamic anisotropy measurements under the TIR conditions.     

Time-resolved luminescence imaging of hydrogen peroxide using sensor membranes in a microwell format

We demonstrate an optical imaging scheme for hydrogen peroxide in a microwell-based format using the europium(III) tetracycline complex as the fluorescent probe, which is incorporated into a polyacrylonitrile-co-polyacrylamide polymer matrix. The resulting sensor membranes are integrated into a 96-microwell plate. Hydrogen peroxide can be visualized by means of time-resolved luminescence lifetime imaging. The imaging system consists of a fast, gated charge-coupled device (CCD) camera and a pulsed array of 96 light emitting diodes (LEDs). Fluorescence lifetime images are acquired in different modes (rapid lifetime determination, RLD, and phase delay rationing, PDR) and compared with conventional intensity-based methods with respect to sensitivity and the dynamic range of the sensor. The lowest limits of detection can be achieved by the RLD method. The response time of the sensor is comparatively high, typically in the range of 10 to 20 minutes, but the response is reversible. The largest signal changes are observed at pH values between 6.5 and 7.5.     

Predicting creep lifetime performance in edgewise compression of containerboards and for stacked corrugated board boxes

Predicting how long time a corrugated board box can be stored with a constant load stacked on top of it without failing is an everyday challenge for a box designer. This is a basic step in corrugated board box material design which is usually resolved by utilizing so-called stacking factors. A stacking factor is the ratio between the compression strength of the box and the stacking load. Higher stacking factors are used to accommodate for longer storing times or high relative humidity conditions. Utilizing stacking factors in the way commonly done today can be a good first approximation. However, this method often neglects or overcompensates for the inherent non-normal distribution statistical behaviour associated with corrugated board box lifetimes. A higher precision in predicting box lifetimes enables material optimization and, thus, a more efficient use of resources. This work presents a convenient way of predicting lifetimes of both containerboards and corrugated board boxes. The approach additionally introduces the concept of reliability into lifetime prediction. The prediction is made directly from material or structural parameters and the applied load. This is accomplished by relating two different parameter sets for the Weibull distribution. It is shown that lifetime is indeed dependent not only on a stacking factor but also on durability and the variations associated with the material or box.     

On the statistical estimation of the lifetimes of charmed particles

Various statistical methods for the estimation of the mean lifetimes of charmed particles have been considered. It is shown that the usual estimates of the maximum likelihood in some cases do not exist or may have rather great positive bias, if the statistics are low. Alternative estimates of the mean lifetime τ are discussed. Among them the “jackknife” estimate of τ is considered. The minimum variance unbiased estimates of the average rate of decay have been derived for some particular cases. It is also shown that the usual estimate of the ratio of the mean lifetimes of different kinds of particles may also have a bias when the statistics are poor. Two methods of reducing this bias have been proposed.     

Time-resolved fluorescence imaging of slab gels for lifetime base-calling in DNA sequencing applications

A compact time-resolved near-IR fluorescence imager was constructed to obtain lifetime and intensity images of DNA sequencing slab gels. The scanner consisted of a microscope body with f/1.2 relay optics onto which was mounted a pulsed diode laser (repetition rate 80 MHz, lasing wavelength 680 nm, average power 5 mW), filtering optics, and a large photoactive area (diameter 500 microns) single-photon avalanche diode that was actively quenched to provide a large dynamic operating range. The time-resolved data were processed using electronics configured in a conventional time-correlated single-photon-counting format with all of the counting hardware situated on a PC card resident on the computer bus. The microscope head produced a timing response of 450 ps (fwhm) in a scanning mode, allowing the measurement of subnano-second lifetimes. The time-resolved microscope head was placed in an automated DNA sequencer and translated across a 21-cm-wide gel plate in approximately 6 s (scan rate 3.5 cm/s) with an accumulation time per pixel of 10 ms. The sampling frequency was 0.17 Hz (duty cycle 0.0017), sufficient to prevent signal aliasing during the electrophoresis separation. Software (written in Visual Basic) allowed acquisition of both the intensity image and lifetime analysis of DNA bands migrating through the gel in real time. Using a dual-labeling (IRD700 and Cy5.5 labeling dyes)/two-lane sequencing strategy, we successfully read 670 bases of a control M13mp18 ssDNA template using lifetime identification. Comparison of the reconstructed sequence with the known sequence of the phage indicated the number of miscalls was only 2, producing an error rate of approximately 0.3% (identification accuracy 99.7%). The lifetimes were calculated using maximum likelihood estimators and allowed on-line determinations with high precision, even when short integration times were used to construct the decay profiles. Comparison of the lifetime base calling to a single-dye/four-lane sequencing strategy indicated similar results in terms of miscalls, but reduced insertion and deletion errors using lifetime identification methods, improving the overall read accuracy.     

In situ time-resolved fluorescence spectroscopy in the frequency domain in capillary electrochromatography

In situ time-resolved fluorescence spectroscopy for capillary electrochromatography (CEC) is described in the frequency domain. Fluorescence decay of the solute molecules is collected directly in the packed stationary phase of the CEC capillary. The fluorescence lifetime profile of the solute molecules reveals the microenvironments they experience in the C18 chromatographic interface. A quartz flow cell and experimental optimization of the signal-to-noise ratio are described that enable the collection of high-quality decay data and subsequent calculation of fluorescence lifetime profiles of the solute molecules. The distribution of pyrene (PY), 1-pyrenemethanol (PY-MeOH), and 1-pyrenebutanol (PY-BuOH) into the C18 stationary phase and the solute-C18 phase interactions are probed, under separation conditions for CEC. All three molecules display a Gaussian distribution of lifetimes, consistent with an ensemble of heterogeneous microenvironments in the C18 stationary phase. The least polar molecule PY diffuses deeply into and interacts extensively with the C18 phase, experiencing high hydrophobicity and significant heterogeneity of microenvironments. The retention order of PY-MeOH, PY-BuOH, and PY in CEC is determined by their interactions with the stationary phase, revealed by their fluorescence lifetime distributions.     

Time resolved spectroscopic studies on some nanophosphors

Time resolved spectroscopy is an important tool for studying photophysical processes in phosphors. Present work investigates the steady state and time resolved photoluminescence (PL) spectroscopic characteristics of ZnS, ZnO and (Zn, Mg)O nanophosphors both in powder as well as thin film form. Photoluminescence (PL) of ZnS nanophosphors typically exhibit a purple/blue emission peak termed as self activated (SA) luminescence and emission at different wavelengths arising due to dopant impurities e.g. green emission for ZnS: Cu, orange emission for ZnS: Mn and red emission for ZnS: Eu. The lifetimes obtained from decay curves range from ns to ms level and suggest the radiative recombination path involving donor-acceptor pair recombination or internal electronic transitions of the impurity atom. A series of ZnMgO nanophosphor thin films with varied Zn: Mg ratios were prepared by chemical bath deposition. Photoluminescence (PL) excitation and emission spectra exhibit variations with changing Mg ratio. Luminescence lifetime as short as 10⁻¹⁰ s was observed for ZnO and ZnMgO (100: 10) nanophosphors. With increasing Mg ratio, PL decay shifts into microsecond range. ZnO and ZnMgO alloys up to 50% Mg were prepared as powder by solid state mixing and sintering at high temperature in reducing atmosphere. Time resolved decay of PL indicated lifetime in the microsecond time scale. The novelty of the work lies in clear experimental evidence of dopants (Cu, Mn, Eu and Mg) in the decay process and luminescence life times in II–VI semiconductor nanocrystals of ZnS and ZnO. For ZnS, blue self activated luminescence decays faster than Cu and Mn related emission. For undoped ZnO nanocrystals, PL decay is in the nanosecond range whereas with Mg doping the decay becomes much slower in the microsecond range.     

Analysis of heterogeneous fluorescence decays. Distribution of pyrene derivatives in an octadecylsilane layer in capillary electrochromatography

The distribution of solute molecules in the stationary phase in capillary electrochromatography (CEC) has been investigated with time-resolved fluorescence in the frequency domain. The analysis of fluorescence decay poses a challenging problem for the complex decay kinetics of heterogeneous systems such as the C18 stationary phase. The nonlinear least-squares (NLLS) method selects the decay model by minimizing the chi2 value. The chi2 criterion, in conjunction with the requirement that the residues should be randomly distributed around zero, frequently leads to a feasible set of multiple decay models that can all fit the data satisfactorily. The maximum entropy method (MEM) further chooses a unique model from the group of feasible ones by maximizing the Shannon-Jaynes entropy. The unique model, however, is not necessarily the most probable one. In this paper, the best model for the fluorescence decays of solute molecules is selected with NLLS using the chi2 statistics, the stability of the fit, and the consistency within replicate experiments. In addition, the recovered lifetime parameters of the true model should display the same trend as the fluorescence decay profiles when an experimental condition is varied. Using these criteria, a Gaussian distribution of fluorescence lifetimes satisfactorily fits the data under all experimental conditions. An additional minor component with a discrete lifetime is attributed to the systematic errors in the measurements. The distribution is a manifestation of an ensemble of heterogeneous microenvironments in the stationary phase of CEC. MEM is not suitable for the modeling of CEC data because of its inaccuracy in recovering broad fluorescence lifetime distributions and its lack of consistency in the replicate measurements in the studies of high-voltage effects.     

Optical properties of Ho:SrMoO4 single crystal

Ho³⁺:SrMoO₄ single crystal was grown by the Czochralski method in N₂ atmosphere. The polarized absorption spectra, emission spectra and the lifetime decay curves were measured at room temperature. The Judd-Ofelt theory has been applied to analyze the absorption spectra. The spectroscopic parameters, including three intensity parameters, radiative transition rates, radiative lifetimes, fluorescent branching ratios and emission cross sections were obtained. The luminescence lifetime of the ⁵S₂ level was determined to be 4.40 μs.     

On-the-fly fluorescence lifetime detection of humic substances in capillary electrophoresis

On-the-fly fluorescence lifetime detection was investigated as a tool for studying humic substances in capillary zone electrophoresis (CZE). Humic substances are complex, heterogeneous mixtures of natural products that tend to migrate in a single, broad CZE peak. The intrinsic fluorescence lifetime of five humic substances from the International Humic Substances Society (IHSS) was monitored using excitation at 488 or 364 nm to produce intensity-lifetime electropherograms for each of the substances. Each frequency-domain lifetime measurement, collected at subsecond intervals during the CZE run, contains the equivalent of a complete decay profile. Lifetime analysis of each decay profile was used to construct a lifetime-resolved electropherogram for each lifetime component, from which the variation in relative intensity contributions of each lifetime across the broad CZE peak could be determined. Absorption spectra, fluorescence excitation-emission spectra, and lifetime profiles of batch solutions of the samples were determined as well. It was found that, whereas absorption and fluorescence spectral characteristics tended to discriminate between humic acids and fulvic acids, the batch solution lifetime profiles discriminated instead between samples from different sources, regardless of fraction. On-the-fly lifetime detection provided a more detailed view of the fluorescence decay of the samples, including greater resolution of lifetimes for two of the fulvic acids and greater discrimination among samples based on lifetime profiles across the CZE peaks.     

Effect of photochemistry on molecular detection by cavity ringdown spectroscopy: case study of an explosive-related compound

Explosives and explosive-related compounds usually have dissociative excited electronic states. We consider the effect of excited-state dissociation upon an absorption event on the UV cavity ringdown spectroscopy (CRDS) detection of these molecules. A change in the photon decay lifetime with increasing laser energy is demonstrated with vapors of 2,6-dinitrotoluene in the open atmosphere. The magnitude of the effect is modeled with coupled equations describing the time-dependent light intensity and molecular concentration within the cavity. The light intensities required within this model to explain the observed changes in the photon decay lifetimes are consistent with the light intensities expected within the cavity under our experimental conditions. It was also found that the slow diffusion of the molecules in static air can magnify the effect of photochemistry on UV CRDS trace detection of molecules with dissociative excited states.     

On-the-Fly Fluorescence Lifetime Determination with Total Emission Detection in HPLC

The entire fluorescence decay profile during HPLC elution has been directly measured on-the-fly in HPLC at higher sensitivity than in previous literature reports. The fluorescence is excited with the fourth harmonic (266 nm) of a pulsed Nd:YAG laser system and detected broadband with a photomultiplier tube and a digital storage oscilloscope. Detection limits in the range 1-10 ppb are found for several individual polycyclic aromatic hydrocarbons (PAHs) when the total time-integrated fluorescence is analyzed. The chromatograms of PAH mixtures containing 8-10 species were lifetime analyzed with a simple phase plane analysis, in which a single lifetime is determined from the fluorescence decay profile for each point on the chromatogram. The determination of lifetimes under coelution conditions is also illustrated and discussed.     

Electromigration in sputtered Al-Cu thin films

Lifetime predictions for aluminum metallization vary considerably depending on many factors. These include the deposition scheme and resulting microstructure of the film, linewidth and geometric variations, alloying elements and testing procedure. Many experimental techniques have been used to study metallization lifetimes. Most involve mass transport measurements in both bulk and thin film samples. A method has been developed for lifetime predictions using a continuous resistance monitoring technique. This allows for lifetime measurements that are more sensitive and, consequently, more accurate than present techniques.     

Single-molecule analysis and lifetime estimates of heterogeneous low-count-rate time-correlated fluorescence data

We demonstrate a proof of concept for detecting heterogeneities and estimating lifetimes in time-correlated single-photon-counting (TCSPC) data when photon counts per molecule are low. In this approach photons are classified as either prompt or delayed according to their arrival times relative to an arbitrarily chosen time gate. Under conditions in which the maximum likelihood (ML) methods fail to distinguish between heterogeneous and homogeneous data sets, histograms of the number of prompt photons from many molecules are analyzed to identify heterogeneities, estimate the contributing fluorescence lifetimes, and determine the relative amplitudes of the fluorescence, scatter, and background components of the signal. The uncertainty of the lifetime estimate is calculated to be larger than but comparable to the uncertainty in ML estimates of single lifetime data made with similar total photon counts. Increased uncertainty and systematic errors in lifetime estimates are observed when the temporal profile of the lifetime decay is similar to either the background or scatter signals, primarily due to error in estimating the amplitudes of the various signal components. Unlike ML methods, which can fail to converge on a solution for a given molecule, this approach does not discard any data, thus reducing the potential for introducing a bias into the results.