Accuracy and precision of manual baseline determination

Vibrational spectra often require baseline removal before further data analysis can be performed. Manual (i.e., user) baseline determination and removal is a common technique used to perform this operation. Currently, little data exists that details the accuracy and precision that can be expected with manual baseline removal techniques. This study addresses this current lack of data. One hundred spectra of varying signal-to-noise ratio (SNR), signal-to-baseline ratio (SBR), baseline slope, and spectral congestion were constructed and baselines were subtracted by 16 volunteers who were categorized as being either experienced or inexperienced in baseline determination. In total, 285 baseline determinations were performed. The general level of accuracy and precision that can be expected for manually determined baselines from spectra of varying SNR, SBR, baseline slope, and spectral congestion is established. Furthermore, the effects of user experience on the accuracy and precision of baseline determination is estimated. The interactions between the above factors in affecting the accuracy and precision of baseline determination is highlighted. Where possible, the functional relationships between accuracy, precision, and the given spectral characteristic are detailed. The results provide users of manual baseline determination useful guidelines in establishing limits of accuracy and precision when performing manual baseline determination, as well as highlighting conditions that confound the accuracy and precision of manual baseline determination.     

Error analysis of the rapid lifetime determination method for double-exponential decays and new windowing schemes

The rapid lifetime determination method (RLD) is a mathematical technique for extremely rapid evaluations of lifetimes in exponential decays. It has been applied in luminescence microscopy and single-molecule lifetime evaluation. To date, the primary application has been in single-exponential evaluations. We present extensions of the method to double exponentials. Using Monte Carlo simulations, we assess the performance of both the double-exponential decay with known lifetimes and the double-exponential decay with unknown preexponential factors and lifetimes. Precision is evaluated as a function of the noise level (Poisson statistics), the ratios of the lifetimes, the ratios of their preexponential factors, and the fitting window. Optimum measurement conditions are determined. RLD is shown to work well over a wide range of practical experimental conditions. If the lifetimes are known, the preexponential factors can be determined with good precision even at low total counts (10(4)). With unknown preexponential factors and lifetimes, precisions decrease but are still acceptable. A new gating scheme (overlapped gating) is shown to offer improved precision for the case of a single-exponential decay. Theoretical predictions are tested against actual experimental data from a laser-based lifetime instrument.     

Four-dimensional multiphoton microscopy with time-correlated single-photon counting

We report on the implementation of fluorescence-lifetime imaging in multiphoton excitation microscopy that uses PC-compatible modules for time-correlated single-photon counting. Four-dimensional data stacks are produced with each pixel featuring fluorescence-decay curves that consist of as many as 4096 bins. Fluorescence lifetime(s) and their amplitude(s) are extracted by statistical methods at each pixel or in arbitrarily defined regions of interest. When employing an avalanche photodiode the width of the temporal response function is 420 ps. Although this response confines the temporal resolution to values greater than several hundreds of picoseconds, the lifetime precision is determined by the signal-to-noise ratio and can be in the range of tens of picosconds. Lifetime changes are visualized in pulsed-laser-deposited fluorescent layers as well as in cyan fluorescent proteins that transfer energy to yellow fluorescent proteins in live mammalian cells.     

Application of Baseline Correction Techniques to the “Ratio Method” of DSC Specific Heat Determination

The measurement of specific heat by heat flux differential scanning calorimetry using the “ratio method” requires three consecutive runs (baseline, reference and sample) to be performed using identical temperature programs. Traditionally measurements are taken as the sample of interest is subjected to a controlled heating rate, but it is also possible to perform measurements on down-ramps, and this is often worthwhile as it provides a consistency check on the up-ramp data. In recent work a Netzsch DSC-404 has been used to investigate discrepancies observed between up- and down-ramp specific heat results. Based upon this experience several different “baseline correction” techniques have been developed, and have been applied to specific heat measurements on reference sapphire samples. The performance of these “baseline correction” techniques is shown to be extremely good, even up to 1400°C. An important conclusion is that, even in cases where agreement between up-ramp and down-ramp data is reasonable, a “baseline correction” method can still usefully be applied to improve the precision of the final results.     

High-precision isothermal titration calorimetry with automated peak-shape analysis

Isothermal titration calorimetry (ITC) is a powerful classical method that enables researchers in many fields to study the thermodynamics of molecular interactions. Primary ITC data comprise the temporal evolution of differential power reporting the heat of reaction during a series of injections of aliquots of a reactant into a sample cell. By integration of each injection peak, an isotherm can be constructed of total changes in enthalpy as a function of changes in solution composition, which is rich in thermodynamic information on the reaction. However, the signals from the injection peaks are superimposed by the stochastically varying time-course of the instrumental baseline power, limiting the precision of ITC isotherms. Here, we describe a method for automated peak assignment based on peak-shape analysis via singular value decomposition in combination with detailed least-squares modeling of local pre- and postinjection baselines. This approach can effectively filter out contributions of short-term noise and adventitious events in the power trace. This method also provides, for the first time, statistical error estimates for the individual isotherm data points. In turn, this results in improved detection limits for high-affinity or low-enthalpy binding reactions and significantly higher precision of the derived thermodynamic parameters.     

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.     

A new approach to MLE of Weibull distribution with interval data

We study the two-parameter maximum likelihood estimation (MLE) problem for the Weibull distribution with consideration of interval data. Without interval data, the problem can be solved easily by regular MLE methods because the restricted MLE of the scale parameter β for a given shape parameter α has an analytical form, thus α can be efficiently solved from its profile score function by traditional numerical methods. In the presence of interval data, however, the analytical form for the restricted MLE of β does not exist and directly applying regular MLE methods could be less efficient and effective. To improve efficiency and effectiveness in handling interval data in the MLE problem, a new approach is developed in this paper. The new approach combines the Weibull-to-exponential transformation technique and the equivalent failure and lifetime technique. The concept of equivalence is developed to estimate exponential failure rates from uncertain data including interval data. Since the definition of equivalent failures and lifetimes follows EM algorithms, convergence of failure rate estimation by applying equivalent failures and lifetimes is mathematically proved. The new approach is demonstrated and validated through two published examples, and its performance in different conditions is studied by Monte Carlo simulations. It indicates that the profile score function for α has only one maximum in most cases. Such good characteristic enables efficient search for the optimal value of α.     

On-chip, time-correlated, fluorescence lifetime extraction algorithms and error analysis

A new, simple, and hardware-only fluorescence-lifetime-imaging microscopy (FLIM) is proposed to implement on-chip lifetime extractions, and their signal-to-noise-ratio based on statistics theory is also deduced. The results are compared with Monte Carlo simulations, giving good agreement. Compared with the commonly used iterative least-squares method or the maximum-likelihood-estimation- (MLE-) based, general purpose FLIM analysis software, our algorithm offers direct calculation of fluorescence lifetime based on the collected photon counts stored in on-chip counters and therefore delivers faster analysis for real-time applications, such as clinical diagnosis. Error analysis considering timing jitter based on statistics theory is carried out for the proposed algorithms and is also compared with MLE to obtain optimized channel width or measurement window and bit resolution of the time-to-digital converters for a given accuracy. A multi-exponential, pipelined fluorescence lifetime method based on the proposed algorithms is also introduced. The performance of the proposed methods has been tested on mono-exponential and four-exponential decay experimental data.     

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.     

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.     

The Weibull log-logistic mixture distributions: Model,theory and application to lifetime data

Lifetime data collected from reliability tests are among data that often exhibit significant heterogeneity caused by variations in manufacturing, which makes standard lifetime models inadequate. Finite mixture models provide more flexibility for modeling such data. In this paper, the Weibull-log-logistic mixture distributions model is introduced as a new class of flexible models for heterogeneous lifetime data. Some statistical properties of the model are presented including the failure rate function, moments generating function, and characteristic function. The identifiability property of the class of all finite mixtures of Weibull-log-logistic distributions is proved. The maximum likelihood estimation (MLE) of model parameters under the Type I and Type II censoring schemes is derived. Some numerical illustrations are performed to study the behavior of the obtained estimators. The model is applied to the hard drive failure data made by the Backblaze data center, where it is found that the proposed model provides more flexibility than the univariate life distributions (Weibull, Exponential, logistic, log-logistic, Frechet). The failure rate of hard disk drives (HDDs) is obtained based on MLE estimates. The analysis of the failure rate function on the basis of SMART attributes shows that the failure of HDDs can have different causes and mechanisms.     

An experimental comparison of the maximum likelihood estimation and nonlinear least-squares fluorescence lifetime analysis of single molecules

Two procedures based on the weighted least-squares (LS) and the maximum likelihood estimation (MLE) method to confidently analyze single-molecule (SM) fluorescence decays with a total number (N) of 2,500-60,000 counts have been elucidated and experimentally compared by analyzing measured bulk and SM decays. The key observation of this comparison is that the LS systematically underestimates the fluorescence lifetimes by approximately 5%, for the range of 1,000-20,000 events, whereas the MLE method gives stable results over the whole intensity range, even at counts N less than 1,000, where the LS analysis delivers unreasonable values. This difference can be attributed to the different statistics approaches and results from improper weighting of the LS method. As expected from theory, the results of both methods become equivalent above a certain threshold of N detected photons per decay, which is here experimentally determined to be approximately 20,000. In contrast to the bulk lifetime distributions, the SM fluorescence lifetime distributions exhibit standard deviations that are sizably larger than the statistically expected values. This comparison proves the strong influence of the inhomogenuous microenvironment on the photophysical behavior of single molecules embedded in a 10-30-nm thin polymer layer.     

A bi-directional flow-rack automated storage and retrieval system for unit-load warehouses

Flow-rack is a multi-deep rack containing multi-row and multi-column slope bins. Traditionally, bins slope in the same direction in a flow-rack to make unit-loads slide from the storage face to the retrieval face driven by gravity, which cause unit-loads are stored to the storage face and retrieved from the retrieval face. In this paper, a bi-directional flow-rack (BFR) is designed, in which bins in adjacent columns slope to opposite directions. In each side of a BFR, unit-loads are stored in half of the bins and retrieved from the other half. Therefore, dual-command (DC) operations could be simultaneously performed on both faces and blocking unit-loads are re-stored to available bins on the same face directly. We develop a travel time model for BFR systems, which provides the throughput baseline for different configurations of BFR. A DC operation generation method is introduced for BFR systems. Simulation experiments are conducted to evaluate the effectiveness of the BFR travel time model, to compare the throughput performance between BFR and SFR systems and to evaluate the performance of the proposed DC operation generation method.     

极大似然判决下大气无线光脉冲位置调制差错性能

大气湍流对无线光通信系统影响较大,基于高斯信道及门限判决方法的大气无线光通信脉冲位置调制(PPM)的误码特性分析存在局限性.针对于此,推导了湍流大气中无线光PPM系统在极大似然判决(MLD)下的差错性能计算模型,并运用蒙特卡洛方法验证了理论计算模型的有效性.仿真分析表明,MLD下PPM具有更好性能,但也较易受湍流影响,为使弱湍流信道中PPM的误比特率不高于10-6,调制阶数每增加1阶,须提高平均接收信噪比0.67 dB以上.对于峰值功率恒定PPM系统,应根据器件性能和具体通信条件,在差错性能和功率利用率之间作出平衡.     

Precision and accuracy of the analog mean-delay method for high-speed fluorescence lifetime measurement

The analog mean-delay (AMD) method is a new alternative method to measure the lifetime of a fluorescence molecule. Because of its powerful advantages of accurate lifetime determination, good photon economy, and a high photon detection rate, the AMD method is considered to be very suitable for high-speed confocal fluorescence lifetime imaging microscopy (FLIM). For the practical usage of the AMD method in FLIM (AMD-FLIM), detailed study on various experimental conditions and parameters that affect the precision and the accuracy of the AMD method is required. In this paper, we present the relation between the precision and accuracy of the lifetime versus iteration number in the AMD method, the best cutoff frequency of a low-pass filter used in the AMD-FLIM system for a given fluorophore, and the optimum position and width of the integration window by using Monte Carlo simulations and a series of AMD-FLIM experiments.     

室内虚拟直线基线的研究

研究了一种室内虚拟直线基线,提出了虚拟直线基线装置建立的原理及采用全站仪进行标定的溯源方法,分析了基线装置的不确定度来源,虚拟直线基线为100 m的距离,估算扩展不确定度U=1.4 mm（k=2）。通过采用该基线装置实际检测手持式激光测距仪,验证了虚拟直线基线的可靠性。     

Calibration approach for fluorescence lifetime determination for applications using time-gated detection and finite pulse width excitation

Time-gated techniques are useful for the rapid sampling of excited-state (fluorescence) emission decays in the time domain. Gated detectors coupled with bright, economical, nanosecond-pulsed light sources like flashlamps and nitrogen lasers are an attractive combination for bioanalytical and biomedical applications. Here we present a calibration approach for lifetime determination that is noniterative and that does not assume a negligible instrument response function (i.e., a negligible excitation pulse width) as does most current rapid lifetime determination approaches. Analogous to a transducer-based sensor, signals from fluorophores of known lifetime (0.5-12 ns) serve as calibration references. A fast avalanche photodiode and a GHz-bandwidth digital oscilloscope is used to detect transient emission from reference samples excited using a nitrogen laser. We find that the normalized time-integrated emission signal is proportional to the lifetime, which can be determined with good reproducibility (typically <100 ps) even for data with poor signal-to-noise ratios ( approximately 20). Results are in good agreement with simulations. Additionally, a new time-gating scheme for fluorescence lifetime imaging applications is proposed. In conclusion, a calibration-based approach is a valuable analysis tool for the rapid determination of lifetime in applications using time-gated detection and finite pulse width excitation.     

Maximum-Likelihood Data Fusion of Phase-Difference and Threshold-Detection Techniques for Wind-Speed Measurement

Analyses of threshold-detection and phase-difference techniques for wind-speed measurement using ultrasonic transducers are presented. The influence of uncertainties that are associated with additive noise and attenuation of the ultrasonic signal on the wind-speed measurement uncertainty is analyzed. A data-fusion procedure based on the maximum-likelihood estimation (MLE) algorithm is developed for the determination of wind speed, with data gathered through threshold-detection and phase-difference techniques. The data-fusion procedure provides a lower measurement uncertainty than those obtained with the above techniques when taken separately. Practical design issues are considered, and an application example is shown to illustrate the proposed procedure.      

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.     

Raman spectra of biological samples: A study of preprocessing methods

In this study preprocessing of Raman spectra of different biological samples has been studied, and their effect on the ability to extract robust and quantitative information has been evaluated. Four data sets of Raman spectra were chosen in order to cover different aspects of biological Raman spectra, and the samples constituted salmon oils, juice samples, salmon meat, and mixtures of fat, protein, and water. A range of frequently used preprocessing methods, as well as combinations of different methods, was evaluated. Different aspects of regression results obtained from partial least squares regression (PLSR) were used as indicators for comparing the effect of different preprocessing methods. The results, as expected, suggest that baseline correction methods should be performed in advance of normalization methods. By performing total intensity normalization after adequate baseline correction, robust calibration models were obtained for all data sets. Combination methods like standard normal variate (SNV), multiplicative signal correction (MSC), and extended multiplicative signal correction (EMSC) in their basic form were not able to handle the baseline features present in several of the data sets, and these methods thus provide no additional benefits compared to the approach of baseline correction in advance of total intensity normalization. EMSC provides additional possibilities that require further investigation.