Instrument Response Function Research Articles

Extraction of lifetime distributions from fluorescence decays with application to DNA-base analogues

Several important aspects of fluorescence decay analysis are addressed and tested against new experimental measurements. A simulated-annealing method is described for deconvoluting the instrument response function from a measured fluorescence decay to yield the true decay, which is more convenient for subsequent fitting. The method is shown to perform well against the conventional approach, which is to fit a convoluted fitting function to the experimentally measured decay. The simulated annealing approach is also successfully applied to the determination of an instrument response function using a known true fluorescence decay (for rhodamine 6G). The analysis of true fluorescence decays is considered critically, focusing specifically on how a distribution of decay constants can be incorporated in to a fit. Various fitting functions are applied to the true fluorescence decays of 2-aminopurine in water-dioxane mixtures, in a dinucleotide, and in DNA duplexes. It is shown how a suitable combination of exponential decays and non-exponential decays (based on a Γ distribution of decay constants) can provide fits of equal quality to the conventional multi-exponential fits used in the majority of previous studies, but with fewer fitting parameters. Crucially, the new approach yields decay-constant distributions that are physically more meaningful than those corresponding to the conventional multi-exponential fit. The methods presented here should find wider application, for example to the analysis of transient-current or optical decays and in Förster resonance energy transfer (FRET).

Precise measurement of protein interacting fractions with fluorescence lifetime imaging microscopy

Precise quantification of endogenous protein-protein interactions across live cells would be a major boon to biology. Such precise measurement is theoretically possible with fluorescence lifetime imaging microscopy (FLIM) but requires first properly addressing multiple biological, instrumental, statistical, and photophysical challenges. We present a detailed investigation of the last three FLIM-specific challenges. Using an efficient, highly accurate analysis code for time-domain FLIM data that accounts for all significant instrumental artifacts (in part, through use of a parametrized model for the instrument response function) and is rigorously based on both conventional statistics (full lifetime histogram fitting by χ(2) minimization) and novel statistics (single pixel fitting of lifetime populations using "maximum fidelity"), we address multiple photophysical challenges, including the proper side-by-side statistical comparison of fluorophore monoexponentiality, the precise assessment of fluorophore lifetimes and lifetime photostability, and the determination of acceptor dark state fractions. Finally, we demonstrate the feasibility of precise measurement of the interacting fraction of a protein across live cells with FLIM.

Use of an improved radiation amplification factor to estimate the effect of total ozone changes on action spectrum weighted irradiances and an instrument response function

Multiple scattering radiative transfer results are used to calculate action spectrum weighted irradiances and fractional irradiance changes in terms of a power law in ozone Ω, U(Ω/200)−RAF, where the new radiation amplification factor (RAF) is just a function of solar zenith angle. Including Rayleigh scattering caused small differences in the estimated 30 year changes in action spectrum‐weighted irradiances compared to estimates that neglect multiple scattering. The radiative transfer results are applied to several action spectra and to an instrument response function corresponding to the Solar Light 501 meter. The effect of changing ozone on two plant damage action spectra are shown for plants with high sensitivity to UVB (280–315 nm) and those with lower sensitivity, showing that the probability for plant damage for the latter has increased since 1979, especially at middle to high latitudes in the Southern Hemisphere. Similarly, there has been an increase in rates of erythemal skin damage and pre‐vitamin D3 production corresponding to measured ozone decreases. An example conversion function is derived to obtain erythemal irradiances and the UV index from measurements with the Solar Light 501 instrument response function. An analytic expressions is given to convert changes in erythemal irradiances to changes in CIE vitamin‐D action spectrum weighted irradiances.

ATMOSPHERIC IMAGING ASSEMBLY MULTITHERMAL LOOP ANALYSIS: FIRST RESULTS

The Atmospheric Imaging Assembly (AIA) on board the Solar Dynamics Observatory has state-of-the-art spatial resolution and shows the most detailed images of coronal loops ever observed. The series of coronal filters peak at different temperatures, which span the range of active regions. These features represent a significant improvement over earlier coronal imagers and make AIA ideal for multithermal analysis. Here, we targeted a 171 A coronal loop in AR 11092 observed by AIA on 2010 August 3. Isothermal analysis using the 171-to-193 ratio gave a temperature of log T ≈ 6.1, similar to the results of Extreme ultraviolet Imaging Spectrograph (EIT) and TRACE. Differential emission measure analysis, however, showed that the plasma was multithermal, not isothermal, with the bulk of the emission measure at log T > 6.1. The result from the isothermal analysis, which is the average of the true plasma distribution weighted by the instrument response functions, appears to be deceptively low. These results have potentially serious implications: EIT and TRACE results, which use the same isothermal method, show substantially smaller temperature gradients than predicted by standard models for loops in hydrodynamic equilibrium and have been used as strong evidence in support of footpoint heating models. These implications may have to be re-examined in the wake of new results from AIA.

Hybrid hard- and soft-modeling approach for the resolution of convoluted femtosecond spectrokinetic data

This work tackles the problem of convolution of femtosecond spectrokinetic data with the instantaneous response function (IRF) of the measurement by hybrid hard- and soft-modeling multivariate curve resolution (MCR). Fitting the time-dependent concentration profiles obtained during the MCR iterative optimization by a kinetic model convoluted with a parametric description of the IRF, the approach benefits of the advantages of both hard- and soft-modeling. The performance of the method is first proven on synthetic data sets with overlap of pure time-dependent concentration and/or spectral profiles and presence of interfering contributions. The method is afterwards applied to the resolution of femtosecond transient absorption spectroscopy data probing ultrafast photoinduced processes implied in the photochromism of anils. An excited-state proton transfer appearing in ~ 80 fs is characterized using an instrumental system with a severe influence of the convoluted instrumental response function, defined by a time resolution of ~ 150 fs.

Effective temporal resolution in pump-probe spectroscopy with strongly chirped pulses

This paper introduces a general theoretical description of femtosecond pump-probe spectroscopy with chirped pulses whose joint spectral and temporal profile is expressed by Wigner spectrograms. We demonstrate that the actual experimental time resolution intimately depends on the pulse-sample interaction and that the commonly used instrumental response function needs to be replaced by a sample-dependent effective response function. We also show that, using the proper configurations in excitation and/or detection, it is possible to overcome the temporal smearing of the measured dynamics due to chirp-induced pulse broadening and recover the temporal resolution that would be afforded by the transform-limited pulses. We verify these predictions with experiments using broadband chirped pump and probe pulses. Our results allow optimization of the temporal resolution in the common case when the chirp of the pump and/or probe pulse is not corrected and may be extended to a broad range of time-resolved experiments.

End-to-End Simulation and Analytical Model of Remote-Sensing Systems: Application to CRISM

The simulation of remote-sensing hyperspectral images is a useful tool for a variety of tasks such as the design of systems, the understanding of the image formation process, and the development and validation of data processing algorithms. The lack of ground truth and the incomplete knowledge of the Martian environment make simulation studies of Mars hyperspectral images a useful tool for automated analysis of Mars data. Hyperspectral near-infrared scenes of mineral mixtures have been simulated to analyze the contributions of surface minerals, atmosphere, and sensor noise on images of Mars. Modeling the remote-sensing process creates a means for the independent analysis of the influence of the environment and instruments on the detection accuracy of the surface composition (e.g., the scene endmembers). The end-to-end model builds surface reflectance scenes based on laboratory sample spectra, creates atmospheric effects using radiative transfer routines, simulates the instrument response function using CRISM data files, and adds instrument noise from thermal and other sources. The purpose of this paper is to understand the hyperspectral remote-sensing process to eventually enable the elevated detection accuracy of minerals on the surface of Mars. The viability of a linear approximation of the complete model is also investigated. The approximation is compared to the complete model in an image classification task.

Time-Resolved Diffuse Optical Spectroscopy: A Differential Absorption Approach

A method is presented for the estimate of spectral changes in the absorption properties of turbid media from time-resolved diffuse optical spectroscopy. The method relies on the hypothesis of constant scattering over the wavelength range of interest, but no limitations come from the sample size and shape as the method is derived directly from the Beer-Lambert law. The effects of a moderate spectral dependence of the scattering properties and of the non-ideal instrument response function were investigated theoretically, and the results were confirmed experimentally, showing that the method can be profitably applied in cases of practical interest.

Implementation of Gold deconvolution for enhanced energy resolution in EEL spectra

Gold’s iterative deconvolution algorithm has been applied to one-dimensional EEL spectra from hexagonal BN. The experimental resolution was varied from 1.1 to 2.25 eV and Gold’s algorithm was able to restore low-loss and core-loss spectra overall well. To estimate the instrument response function, the most convenient method was to extract the zero-loss peak from the low-loss spectrum. By instead using low-loss spectra as kernel, as suggested by Egerton, enhanced energy resolution could also be obtained with plural scattering simultaneously removed. It is further shown how the FWHM of the π ⁎ peak in the boron K-edge of hexagonal BN is reduced from 1.4 to 0.7 eV with almost no noise amplification after 500 iterations while resolving the σ ⁎ doublet. The result was almost identical after a stunning 5000–10,000 iterations, implying that Gold’s method converges and can be stable for a large number of iterations. However, for lower-intensity spectra the number of iterations is limited. The results close to the intense zero-loss peak were uncertain and further testing with better experimental resolution is recommended. It is also found that to improve the resolution and not just sharpen the spectra, a large number of iterations is required.

Mapping Depth to Argillic Soil Horizons Using Apparent Electrical Conductivity

Maps of apparent electrical conductivity [Formula: see text] of the soil profile are widely used in precision agriculture. A number of [Formula: see text] sensors are commercially available, each with a unique response function (i.e., the relative contribution of soil at each depth to the integrated [Formula: see text] reading). Our past research estimated depth to an argillic horizon (i.e., topsoil depth, TD) on claypan soils by fitting empirical equations to [Formula: see text] sensor data. The objective of this research was to determine if TD estimates could be improved by combining data from multiple [Formula: see text] sensors and by solving for TD by inverting a two-layer soil model incorporating instrument response functions. Data were obtained with three sensors having five different [Formula: see text] depth-response functions (Veris 3150 (mention of trade names or commercial products is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture or the University of Florida), Geonics EM38 vertical dipole mode, and DUALEM-2S) on two Missouri claypan-soil fields. Soil cores obtained in each field provided measured TD data for calibration and validation. Using a numerical optimization approach, response-function models were developed for [Formula: see text] variables individually and in combination. Similarly, linear regression was applied to single and multiple variables. Root mean square error of validation [Formula: see text] of single-variable TD estimates was [Formula: see text], with better results for those variables with moderately deep [Formula: see text] response functions. Results from the model-based approach were very similar to those obtained by regressing TD on [Formula: see text]. The best calibrations using multiple variables in model inversion or regression were somewhat better than those using single variables, with [Formula: see text] of [Formula: see text] and [Formula: see text], respectively. For all approaches, highest TD errors were localized to one area of one field, possibly because soils in this area violated the model assumption of spatially homogeneous soil layer conductivity. Although these calibrations are sufficiently accurate to be useful in TD mapping, a model solution allowing layer conductivities to vary spatially should be investigated for possible improvements.

Self-quenching of uranin: Instrument response function for color sensitive photo-detectors.

Concentration is a key determining factor in the fluorescence properties of organic fluorophores. We studied self-quenching of disodium fluorescein (uranin) fluorescence in polyvinyl alcohol (PVA) thin films. The concentration dependent changes in brightness and anisotropy were followed by a lifetime decrease. We found that at a concentration of 0.54 M, the lifetime decreases to 7 ps. At a concentration of 0.18 M the lifetime was 10 ps with the relatively high quantum yield of 0.002. In these conditions the fluorescence intensity decay was homogeneous (well approximated by a single lifetime). We realized that such a sample was an ideal fluorescence lifetime standard for spectroscopy and microscopy, and therefore characterized instrument response functions for a time-domain technique. We show that self-quenched uranin enables measurements free of the color effect, making it a superior choice for a lifetime reference over scattered light.

HANDLING DRAWBACKS OF MASS SPECTROMETRIC DETECTION COUPLED TO LIQUID CHROMATOGRAPHY IN BIOANALYSIS

Hyphenation of mass spectrometry (MS) to liquid chromatography (LC) represents a powerful tool for qualitative and quantitative characterization of target compounds in very complex matrixes of biological origins. In spite of many advantages due to recent advances and innovations in the area of instrumentation and dedicated software support, some difficulties are still encountered in its current applications. The large variety of functional principles and technical solutions applied for hyphenation of the two techniques, for ion sources, ion extraction and focusing, mass analysis, and ion counting makes it more difficult to obtain perfect agreement between the intrinsic characteristics of the laboratory-available instrumentation and the declared goals of specific determination. This review covers a part of the literature data dealing with the shortcomings of LC/MS in bioanalysis. The following topics are discussed: structural identification and confirmation in LC/MS; precision of the instrumental response over short and long term periods; non-linear response functions; adduct formation in atmospheric pressure ion sources; and carryover effects. Most of the problems arising in LC/MS are related to phenomena occurring during ionization. Obviously, the structural characteristics of the analyzed compounds play an important role, although the principles of ionization within the source and the supporting technical solutions and constructive designs add their own particular features. The complex influence of residual sample matrixes over ionization yields of target compounds and internal standards needs to be studied through proper experimental procedures, in order to control both precision and instrumental response function in analysis of biological samples.

Deconvolution of femtosecond time‐resolved spectroscopy data in multivariate curve resolution. Application to the characterization of ultrafast photo‐induced intramolecular proton transfer

AbstractIn femtosecond absorption spectroscopy, deconvolution of the measured kinetic traces is still an important issue as photochemical processes that may possess shorter characteristic times than the time resolution of the experiment are usually considered. In this work, we propose to perform deconvolution of the time‐dependent concentration profiles extracted from multivariate curve resolution (MCR) applied to spectrokinetic data. The profiles are fitted with a model function including a description of the instrumental response function (IRF) of the experiment. The method combines the potential benefits of soft‐modeling data analysis with the ones of hard‐modeling for parameter estimation. The potential of the method is demonstrated first analyzing five synthetic data sets for which IRF of different widths are simulated. It is then successfully applied to resolve femtosecond UV‐visible transient absorption spectroscopy data investigating the photodynamics of salicylidene aniline, a photochromic molecule of wide interest. Considering a time resolution of 150 fs for the IRF, a characteristic time of 45 fs is recovered for the first step of the photo‐induced process which consists of an ultrafast intramolecular proton transfer. Our results also confirm the existence of an intermediate species with a characteristic time of 240 fs. Copyright © 2010 John Wiley & Sons, Ltd.

Deconvolution of u channel magnetometer data: Experimental study of accuracy, resolution, and stability of different inversion methods

We explore the effects of sampling density, signal/noise ratios, and position‐dependent measurement errors on deconvolution calculations for u channel magnetometer data, using a combination of experimental and numerical approaches. Experiments involve a synthetic sample set made by setting hydraulic cement in a 30‐cm u channel and slicing the hardened material into ∼2‐cm lengths, and a natural lake sediment u channel sample. The cement segments can be magnetized and measured individually, and reassembled for continuous u channel measurement and deconvolution; the lake sediment channel was first measured continuously and then sliced into discrete samples for individual measurement. Each continuous data set was deconvolved using the ABIC minimization code of Oda and Shibuya (1996) and two new approaches that we have developed, using singular‐value decomposition and regularized least squares. These involve somewhat different methods to stabilize the inverse calculations and different criteria for identifying the optimum solution, but we find in all of our experiments that the three methods converge to essentially identical solutions. Repeat scans in several experiments show that measurement errors are not distributed with position‐independent variance; errors in setting/determining the u channel position (standard deviation ∼0.2 mm) translate in regions of strong gradients into measurement uncertainties much larger than those due to instrument noise and drift. When we incorporate these depth‐dependent measurement uncertainties into the deconvolution calculations, the resulting models show decreased stability and accuracy compared to inversions assuming depth‐independent measurement errors. The cement experiments involved varying directions and uniform intensities downcore, and very good accuracy was obtained using all of the methods when the signal/noise ratio was greater than a few hundred and the sampling interval no larger than half the length scale of magnetization changes. Addition of synthetic noise or reduction of sampling density decreased the resolution and accuracy of all the methods equally. The sediment‐core experiment involved uniform (axial) magnetization direction and strongly varying intensities downcore. Intensity variations are well resolved and directions are accurate to within about 5 degrees, with errors attributable to omission and/or inaccurate calibration of cross terms in the instrument response function.

Photophysical properties of novel fluorescein derivative and its applications for time-resolved fluorescence spectroscopy

We propose a new FRET-based fluorescent molecule that can be used to collect instrument response function. We used 5-carboxyfluorescein as a donor and BHQ2 as an acceptor. The decrease of lifetime was from 4.00 ns to 18 ps for new compound. The brightness of Fl-BHQ2 is two to three orders of magnitude stronger than Raman signal. The solution of Fl-BHQ2 can be used to characterize temporal responses of photodetectors used in spectroscopy/microscopy. We used Fl-BHQ2 fluorescence as a reference in time-domain measurements and we got the same or even better results as those with light scattering.

Reference optical phantoms for diffuse optical spectroscopy Part 1 – Error analysis of a time resolved transmittance characterization method

Development, production quality control and calibration of optical tissue-mimicking phantoms require a convenient and robust characterization method with known absolute accuracy. We present a solid phantom characterization technique based on time resolved transmittance measurement of light through a relatively small phantom sample. The small size of the sample enables characterization of every material batch produced in a routine phantoms production. Time resolved transmittance data are pre-processed to correct for dark noise, sample thickness and instrument response function. Pre-processed data are then compared to a forward model based on the radiative transfer equation solved through Monte Carlo simulations accurately taking into account the finite geometry of the sample. The computational burden of the Monte-Carlo technique was alleviated by building a lookup table of pre-computed results and using interpolation to obtain modeled transmittance traces at intermediate values of the optical properties. Near perfect fit residuals are obtained with a fit window using all data above 1% of the maximum value of the time resolved transmittance trace. Absolute accuracy of the method is estimated through a thorough error analysis which takes into account the following contributions: measurement noise, system repeatability, instrument response function stability, sample thickness variation refractive index inaccuracy, time correlated single photon counting system time based inaccuracy and forward model inaccuracy. Two sigma absolute error estimates of 0.01 cm(-1) (11.3%) and 0.67 cm(-1) (6.8%) are obtained for the absorption coefficient and reduced scattering coefficient respectively.

Fluorescence Spectroscopic Studies of (Acetamide + Sodium/Potassium Thiocyanates) Molten Mixtures: Composition and Temperature Dependence

Steady state and time-resolved fluorescence spectroscopic techniques have been used to explore the Stokes' shift dynamics and rotational relaxation of a dipolar solute probe in molten mixtures of acetamide (CH(3)CONH(2)) with sodium and potassium thiocyanates (Na /KSCN) at T approximately 318 K and several other higher temperatures. The dipolar solute probe employed for this study is coumarin 153 (C153). Six different fractions (f) of KSCN of the following ternary mixture composition, 0.75 CH(3)CONH(2) + 0.25[(1 - f)NaSCN + fKSCN], have been considered. The estimated experimental dynamic Stokes' shift for these systems ranges between 1800 and 2200 cm(-1) (+/-250 cm(-1)), which is similar to what has been observed with the same solute probe in several imidazolium cation based room temperature ionic liquids (RTIL) and in pure amide solvents. Interestingly, this range of estimated Stokes' shift, even though not corresponding to the megavalue of static dielectric constant reported in the literature for a binary mixture of molten CH(3)CONH(2) and NaSCN, exhibits a nonmonotonic KSCN concentration dependence. The magnitudes of the dynamic Stokes' shift detected in the present experiments are significantly less than the estimated ones, as nearly 40-60% of the total shift is missed due to the limited time resolution employed (full-width at half-maximum of the instrument response function approximately 70 ps). The solvation response function, constructed from the detected shifts in these systems, exhibits triexponential decay with the fastest time constant (tau(1)) in the 10-20 ps range, which might be much shorter if measured with a better time resolution. The second time constant (tau(2)) lies in the 70-100 ps range, and the third one (tau(3)) ranges between 300 and 800 ps. Both these time constants (tau(2) and tau(3)) show alkali metal ion concentration dependence and exhibit viscosity decoupling at higher viscosity in the NaSCN-enriched region. Time dependent rotational anisotropy has been found to be biexponential at all mixture compositions studied. Both the average solvation (<tau(s)>) and rotation (<tau(r)>) times of C153 in these mixtures exhibit fractional power law dependence on medium viscosity (<tau(x)> is proportional to eta(p), x being solvation or rotation). For solvation, p is found to be 0.46, which is very different from that obtained for common polar and nonpolar solvents, and RTILs (p approximately = 1). For rotation, p approximately = 0.65, which is again different from the value (p approximately = 1) obtained for common polar solvents and RTILs but very similar to that (p approximately = 0.6) found for nonpolar solvents. In addition, experimentally measured average rotation times in these mixtures are found to exhibit slip behavior in the low eta/T region, which gradually transforms to subslip as eta/T increases. Calculations using a recently developed semimolecular theory predict a total dynamic Stokes' shift for C153 (dipolar solute) in these molten mixtures near approximately 1600 cm(-1) where the solute-solvent (dipole-dipole) and the ion-solute (ion-dipole) interactions contribute respectively approximately 80% and approximately 20% to the calculated total shift. Like in experiments, the theoretically predicted solvation response function in the overdamped limit at each mixture composition has been found to be triexponential. The calculations in the underdamped limit, however, suggest a biphasic decay where a composition independent subpicosecond component and a much slower component with the time constant spreading over 150-850 ps contribute equally to constitute the total decay. The calculated average solvation times in this limit are found to be in better agreement with experimental results than the predictions from the overdamped limit.

Determining the sea-air flux of dimethylsulfide by eddy correlation using mass spectrometry

Abstract. Mass spectrometric measurement of DMS by atmospheric pressure ionization with an isotopically labeled standard (APIMS-ILS) is a sensitive method with sufficient bandpass for direct flux measurements by eddy correlation. Use of an isotopically labeled internal standard greatly reduces instrumental drift, improving accuracy and precision. APIMS-ILS has been used in several recent campaigns to study ocean-atmosphere gas transfer and the chemical budget of DMS in the marine boundary layer. This paper provides a comprehensive description of the method and errors associated with DMS flux measurement from ship platforms. The APIMS-ILS instrument used by most groups today has a sensitivity of 100–200 counts s−1 pptv−1, which is shown to be more than sufficient for flux measurement by eddy covariance. Mass spectral backgrounds (blanks) are determined by stripping DMS from ambient air with gold. The instrument is found to exhibit some high frequency signal loss, with a half-power frequency of ≈1 Hz, but a correction based on an empirically determined instrument response function is presented. Standard micrometeorological assumptions of steady state and horizontal uniformity are found to be appropriate for DMS flux measurement, but rapid changes in mean DMS mixing ratio may serve as a warning that measured flux does not represent the true surface flux. In addition, bias in surface flux estimates arising from the flux divergence is not generally significant in the surface layer, but under conditions of lowered inversion and high flux may become so. The effects of error in motion corrections and of vertical motion within the surface layer concentration gradient are discussed and the estimated maximum error from these effects is ≤18%.

Technique to calculate geometric spontaneous emission factor from amplified spontaneous emission spectrum of FP semiconductor laser

The geometric spontaneous emission factor is calculated from the amplified spontaneous emission spectrum of a Fabry-Pérot (FP) laser diode. The optical loss and quasi-Fermi level separation are determined first using the Fourier transform method with the influence from the instrument response function being taken into account. From this the geometric spontaneous emission factor is calculated to be 5.95×10−3 just below laser threshold.

Phase transition kinetics of lipid bilayer membranes studied by time-resolved pressure perturbation calorimetry

The relaxation kinetics of aqueous lipid dispersions after a pressure jump (p-jump) was investigated using time-resolved pressure perturbation calorimetry (PPC). Analysis of the calorimetric response curves by deconvolution with the instrumental response function gives information about slow processes connected with the lipid phase transition. The lipid transition from the gel to the liquid-crystalline state was found to be a multi-step process with relaxation constants in the seconds range resolvable by time-resolved PPC and faster processes with relaxation times shorter than ca. 5 s that could not be resolved by the instrument. The faster processes comprise ca. 50% of the total heat uptake at the transition midpoint. This is the first calorimetric measurement showing the multi-step nature of the transition. The results are in good agreement with data obtained with other detection methods and with molecular modelling experiments describing the transition as a multi-step process with nucleation and growth steps.