Instrument Response Function Research Articles

Lifetime-based analysis of binary fluorophores mixtures in the low photon count limit

SummarySingle biomolecule sensing often requires the quantification of multiple fluorescent species. Here, we theoretically and experimentally use time-resolved fluorescence via Time Correlated Single Photon Counting (TCSPC) to accurately quantify fluorescent species with similar chromatic signatures. A modified maximum likelihood estimator is introduced to include two fluorophore species, with compensation of the instrument response function. We apply this algorithm to simulated data of a simplified two-fluorescent species model, as well as to experimental data of fluorophores' mixtures and to a model protein, doubly labeled with different fluorophores' ratio. We show that 100 to 200 photons per fluorophore, in a 10-ms timescale, are sufficient to provide an accurate estimation of the dyes' ratio on the model protein. Our results provide estimation for the desired photon integration time toward implementation of TCSPC in systems with fast occurring events, such as translocation of biomolecules through nanopores or single-molecule burst analyses experiments.

Calibration of the Marshall Grazing Incidence X-Ray Spectrometer Experiment. II. Flight Instrument Calibration

Abstract The Marshall Grazing Incidence X-ray Spectrometer (MaGIXS) is a sounding rocket experiment that observes the soft X-ray spectrum of the Sun from 6.0–24 Å (0.5–2.0 keV), successfully launched on 2021 July 30. End-to-end alignment of the flight instrument and calibration experiments are carried out using the X-ray and Cryogenic Facility at NASA Marshall Space Flight Center. In this paper, we present the calibration experiments of MaGIXS, which include wavelength calibration, measurement of line spread function, and determination of effective area. Finally, we use the measured instrument response function to predict the expected count rates for MaGIXS flight observation looking at a typical solar active region.

Calibration of the temporal drift in absolute and relative Raman intensities in large Raman images using a mercury–argon lamp

AbstractRaman imaging is a powerful tool for the analysis of astromaterials including meteorites, cosmic dust, and samples returned to Earth by spacecraft. Many astromaterials are heterogeneous on mm‐ or cm‐sized scales requiring the collection of large Raman images with long accumulation times of days to weeks. It is known that the wavenumber and bandwidth calibration of a Raman instrument drifts with time and temperature resulting in significant changes in the observed Raman band over the course of Raman image collection. In this work, we examine the temporal and thermal drift of absolute and relative Raman intensities through the collection of a Raman image. We show that the spectrometer throughput and observed Raman intensities change over time and are correlated to small changes in lab temperature. We propose a method of calibrating for the drift in Raman intensities using a Hg–Ar (mercury–argon) calibration lamp integrated into the Raman instrument. We also show that relative Raman intensities drift with time as a function of temperature as a result of changes to the Raman instrument response function. We developed a method to calibrate the drifting instrument response function using only the calibration lines from the Hg–Ar lamp. Overall, our results elucidate the effects of temporal/thermal drift on Raman intensities and we propose calibration techniques to account for these effects.

Vibronic dynamics resolved by global and target analysis of ultrafast transient absorption spectra.

We present a methodology that provides a complete parametric description of the time evolution of the electronically and vibrationally excited states as detected by ultrafast transient absorption (TA). Differently from previous approaches, which started fitting the data after ≈100fs, no data are left out in our methodology, and the "coherent artifact" and the instrument response function are fully taken into account. In case studies, the method is applied to solvents, the dye Nile blue, and all-trans β-carotene in cyclohexane solution. The estimated Damped Oscillation Associated Spectra (DOAS) and phases express the most important vibrational frequencies present in the molecular system. By global fit alone of the experimental data, it is difficult to interpret in detail the underlying dynamics. Since it is unfeasible to directly fit the data by a theoretical simulation, our enhanced DOAS methodology thus provides a useful "middle ground" where the theoretical description and the fit of the experimental data can meet. β-carotene in cyclohexane was complementarily studied with femtosecond stimulated Raman spectroscopy (FSRS). The fs-ps dynamics of β-carotene in cyclohexane in TA and FSRS experiments can be described by a sequential scheme S2 → hot S1 → S1' → S1 → S0 with lifetimes of 167fs (fixed), 0.35, 1.1, and 9.6ps. The correspondence of DOAS decaying concomitantly with hot S1 and the Species Associated Difference Spectra of hot S1 in TA and FSRS suggest that we observe here features of the vibrational relaxation and nuclear reorganization responsible for the hot S1 to S1 transition.

Estimation of electron fluxes in the Earth's geostationary orbit with Tikhonov regularization during a magnetically quiet period

Electron energy fluxes in the Earth's outer radiation belt are estimated using an inverse theory of the Tikhonov regularization based upon observations in geostationary orbits. Particle Detector (PD) experiment aboard a geostationary satellite GEO-KOMPSAT-2A (GK2A) at a geographic longitude of 128.2°E provided observations of electrons within a 150–2,400 keV energy range with an unprecedented energy resolution of ΔE/E in the range of 5–25%. Instrument response functions, calculated with Monte-Carlo simulations, are deconvoluted with electron observations. Using regularization parameters determined from Generalized Cross Validation (GCV), the Tikhonov method was applied to observations made during a geomagnetically quiet period. This Tikhonov regularization method, now possible for observations in the Earth's radiation belt for the first time, allows direct inference of electron fluxes without resorting to predetermined functional forms. Comparisons of our results with those from conventional methods indicate differences among the results as large as ∼200%.

Multivariate Curve Resolution Slicing of Multiexponential Time-Resolved Spectroscopy Fluorescence Data.

Time-resolved fluorescence spectroscopy (TRFS), i.e., measurement of fluorescence decay curves for different excitation and/or emission wavelengths, provides specific and sensitive local information on molecules and on their environment. However, TRFS relies on multiexponential data fitting to derive fluorescence lifetimes from the measured decay curves and the time resolution of the technique is limited by the instrumental response function (IRF). We propose here a multivariate curve resolution (MCR) approach based on data slicing to perform tailored and fit-free analysis of multiexponential fluorescence decay curves. MCR slicing, taking as a basic framework the multivariate curve resolution-alternating least-squares (MCR-ALS) soft-modeling algorithm, relies on a hybrid bilinear/trilinear data decomposition. A key feature of the method is that it enables the recovery of individual components characterized by decay profiles that are only partially describable by monoexponential functions. For TRFS data, not only pure multiexponential tail information but also shorter time delay information can be decomposed, where the signal deviates from the ideal exponential behavior due to the limited time resolution. The accuracy of the proposed approach is validated by analyzing mixtures of three commercial dyes and characterizing the mixture composition, lifetimes, and associated contributions, even in situations where only ternary mixture samples are available. MCR slicing is also applied to the analysis of TRFS data obtained on a photoswitchable fluorescent protein (rsEGFP2). Three fluorescence lifetimes are extracted, along with the profile of the IRF, highlighting that decomposition of complex systems, for which individual isomers are characterized by different exponential decays, can also be achieved.

Ultrafast-laser-absorption spectroscopy in the mid-infrared for single-shot, calibration-free temperature and species measurements in low- and high-pressure combustion gases.

This manuscript presents an ultrafast-laser-absorption-spectroscopy (ULAS) diagnostic capable of providing calibration-free, single-shot measurements of temperature and CO at 5 kHz in combustion gases at low and high pressures. Additionally, this diagnostic was extended to provide 1D, single-shot measurements of temperature and CO in a propellant flame. A detailed description of the spectral-fitting routine, data-processing procedures, and determination of the instrument response function are also presented. The accuracy of the diagnostic was validated at 1000 K and pressures up to 40 bar in a heated-gas cell before being applied to characterize the spatiotemporal evolution of temperature and CO in AP-HTPB and AP-HTPB-aluminum propellant flames at pressures between 1 and 40 bar. The results presented here demonstrate that ULAS in the mid-IR can provide high-fidelity, calibration-free measurements of gas properties with sub-nanosecond time resolution in harsh, high-pressure combustion environments representative of rocket motors.

Optimization of time domain diffuse correlation spectroscopy parameters for measuring brain blood flow.

.Significance: Time domain diffuse correlation spectroscopy (TD-DCS) can offer increased sensitivity to cerebral hemodynamics and reduced contamination from extracerebral layers by differentiating photons based on their travel time in tissue. We have developed rigorous simulation and evaluation procedures to determine the optimal time gate parameters for monitoring cerebral perfusion considering instrumentation characteristics and realistic measurement noise.Aim: We simulate TD-DCS cerebral perfusion monitoring performance for different instrument response functions (IRFs) in the presence of realistic experimental noise and evaluate metrics of sensitivity to brain blood flow, signal-to-noise ratio (SNR), and ability to reject the influence of extracerebral blood flow across a variety of time gates to determine optimal operating parameters.Approach: Light propagation was modeled on an MRI-derived human head geometry using Monte Carlo simulations for 765- and 1064-nm excitation wavelengths. We use a virtual probe with a source–detector separation of 1 cm placed in the pre-frontal region. Performance metrics described above were evaluated to determine optimal time gate(s) for different IRFs. Validation of simulation noise estimates was done with experiments conducted on an intralipid-based liquid phantom.Results: We find that TD-DCS performance strongly depends on the system IRF. Among Gaussian pulse shapes, pulse length appears to offer the best performance, at wide gates (500 ps and larger) with start times 400 and 600 ps after the peak of the TPSF at 765 and 1064 nm, respectively, for a 1-s integration time at photon detection rates seen experimentally (600 kcps at 765 nm and 4 Mcps at 1064 nm).Conclusions: Our work shows that optimal time gates satisfy competing requirements for sufficient sensitivity and sufficient SNR. The achievable performance is further impacted by system IRF with quasi-Gaussian pulse obtained using electro-optic laser shaping providing the best results.

Performance assessment of laser sources for time-domain diffuse correlation spectroscopy.

Time-domain diffuse correlation spectroscopy (TD-DCS) is an emerging optical technique that enables noninvasive measurement of microvascular blood flow with photon path-length resolution. In TD-DCS, a picosecond pulsed laser with a long coherence length, adequate illumination power, and narrow instrument response function (IRF) is required, and satisfying all these features is challenging. To this purpose, in this study we characterized the performance of three different laser sources for TD-DCS. First, the sources were evaluated based on their emission spectrum and IRF. Then, we compared the signal-to-noise ratio and the sensitivity to velocity changes of scattering particles in a series of phantom measurements. We also compared the results for in vivo measurements, performing an arterial occlusion protocol on the forearm of three adult subjects. Overall, each laser has the potential to be successfully used both for laboratory and clinical applications. However, we found that the effects caused by the IRF are more significant than the effect of a limited temporal coherence.

A Proposal for Relative In-flight Flux Self-calibrations for Spectro-photometric Surveys

We present a method for the in-flight relative flux self-calibration of a spectro-photometer instrument, general enough to be applied to any upcoming galaxy survey on satellite. The instrument response function, that accounts for a smooth continuous variation due to telescope optics, on top of a discontinuous effect due to the segmentation of the detector, is inferred with a χ 2 statistics. The method provides unbiased inference of the sources count rates and of the reconstructed relative response function, in the limit of high count rates. We simulate a simplified sequence of observations following a spatial random pattern and realistic distributions of sources and count rates, with the purpose of quantifying the relative importance of the number of sources and exposures for correctly reconstructing the instrument response. We present a validation of the method, with the definition of figures of merit to quantify the expected performance, in plausible scenarios.

Biochemical resolving power of fluorescence lifetime imaging: untangling the roles of the instrument response function and photon-statistics.

A deeper understanding of spatial resolution has led to innovations in microscopy and the disruption of biomedical research, as with super-resolution microscopy. To foster similar advances in time-resolved and spectral imaging, we have previously introduced the concept of ‘biochemical resolving power’ in fluorescence microscopy. Here, we apply those concepts to investigate how the instrument response function (IRF), sampling conditions, and photon-statistics limit the biochemical resolution of fluorescence lifetime microscopy. Using Fisher information analysis and Monte Carlo simulations, we reveal the complex dependencies between photon-statistics and the IRF, permitting us to quantify resolution limits that have been poorly understood (e.g., the minimum resolvable decay time for a given width of the IRF and photon-statistics) or previously underappreciated (e.g., optimization of the IRF for biochemical detection). With this work, we unravel common misunderstandings on the role of the IRF and provide theoretical insights with significant practical implications on the design and use of time-resolved instrumentation.

Some aspects about time broadening in fluorescence up-conversion measurements.

Several aspects contributing to the temporal broadening in the measurement of ultrafast fluorescence by means of up-conversion wave mixing are presented: the characteristics of the sample, those of the collection optics, and the wave mixing with the gate pulse in a non-linear crystal. It is concluded that these contributions are emission wavelength dependent and can be as important as the pulse durations in determining the instrument response function in this technique.

Single-Photon Detection Module Based on Large-Area Silicon Photomultipliers for Time-Domain Diffuse Optics

Silicon photomultipliers (SiPM) are pixelated single-photon detectors combining high sensitivity, good time resolution and high dynamic range. They are emerging in many fields, such as time-domain diffuse optics (TD-DO). This is a promising technique in neurology, oncology, and quality assessment of food, wood, and pharmaceuticals. SiPMs can have very large areas and can significantly increase the sensitivity of TD-DO in tissue investigation. However, such improvement is currently limited by the high detector noise and the worsening of SiPM single-photon time resolution due to the large parasitic capacitances. To overcome such limitation, in this paper, we present two single-photon detection modules, based on 6 × 6 mm2 and 10 × 10 mm2 SiPMs, housed in vacuum-sealed TO packages, cooled to −15 °C and −36 °C, respectively. They integrate front-end amplifiers and temperature controllers, being very useful instruments for TD-DO and other biological and physical applications. The signal extraction from the SiPM was improved. The noise is reduced by more than two orders of magnitude compared to the room temperature level. The full suitability of the proposed detectors for TD-DO measurements is outside the scope of this work, but preliminary tests were performed analyzing the shape and the stability of the Instrument Response Function. The proposed modules are thus fundamental building blocks to push the TD-DO towards deeper investigations inside the body.

Water and Carbon Dioxide Adsorption on CaO(001) Studied via Single Crystal Adsorption Calorimetry

A new method to analyze microcalorimetry data was employed to study the adsorption energies and sticking probabilities of D2O and CO2 on CaO(001) at several temperatures. This method deconvolutes the line shapes of the heat detector response into an instrument response function and exponential decay functions, which correspond to the desorption of distinct surface species. This allows for a thorough analysis of the adsorption, dissociation, and desorption processes that occur during our microcalorimetry experiments. Our microcalorimetry results, show that D2O adsorbs initially with an adsorption energy of 85–90 kJ/mol at temperatures ranging from 120 to 300 K, consistent with prior spectroscopic studies that indicate dissociation. This adsorption energy decreases with increasing coverage until either D2O multilayers are formed at low temperatures (120 K) or the surface is saturated (> 150 K). Artificially producing defects on the surface by sputtering prior to dosing D2O sharply increases this adsorption energy, but these defects may be healed after annealing the surface to 1300 K. CO2 adsorbs on CaO(001) with an initial adsorption energy of ~ 125 kJ/mol, and decreases until the saturation coverage is reached, which is a function of surface temperature. The results showed that pre-adsorbed water blocks adsorption sites, lowers the saturation coverage, and lowers the measured adsorption energy of CO2. The calorimetry data further adds to our understanding of D2O and CO2 adsorption on oxide surfaces.

Application of an energy-dependent instrument response function to analysis of nTOF data from cryogenic DT experiments

Neutron time-of-flight (nTOF) detectors are used to diagnose the conditions present in inertial confinement fusion (ICF) experiments and basic laboratory physics experiments performed on an ICF platform. The instrument response function (IRF) of these detectors is constructed by convolution of two components: an x-ray IRF and a neutron interaction response. The shape of the neutron interaction response varies with incident neutron energy, changing the shape of the total IRF. Analyses of nTOF data that span a broad range of energies must account for this energy-dependence in order to accurately infer plasma parameters and nuclear properties in ICF experiments. This work briefly reviews a matrix multiplication approach to convolution, which allows for an energy-dependent change in the shape of the IRF. This method is applied to synthetic data resembling symmetric cryogenic DT implosions to examine the effect of the energy-dependent IRF on the inferred areal density. The results of forward fits that infer ion temperatures and areal densities from nTOF data collected during cryogenic DT experiments on OMEGA are also discussed.

Proof-of-concept of a neutron time-of-flight ellipsoidal detector.

The time-resolved measurement of neutrons emitted from nuclear implosions at inertial confinement fusion facilities is used to characterize the fusing plasma. Several significant quantities are routinely measured by neutron time-of-flight (nToF) detectors in these experiments. Current nToF detectors use scintillators as well as solid-state Cherenkov radiators. The latter has an inherently faster time response and can provide a co-registered γ-ray measurement as well as improved precision in the bulk hot-spot velocity. This work discusses a nToF ellipsoidal detector that also utilizes a solid-state Cherenkov radiator. The detector has the potential to achieve a fast instrument response function allowing for characterization of the γ-ray burn history as well as the ability to field the detector closer to the fusion source. Proof-of-concept testing of the nToF ellipsoidal detector has been conducted at the National Ignition Facility using commercial optics. A time-resolved neutron signal has been measured from the diagnostic. Preliminary simulations corroborate the results.

Accuracy of homogeneous models for photon diffusion in estimating neonatal cerebral hemodynamics by TD-NIRS.

We assessed the accuracy of homogenous (semi-infinite, spherical) photon diffusion models in estimating absolute hemodynamic parameters of the neonatal brain in realistic scenarios (ischemia, hyperoxygenation, and hypoventilation) from 1.5 cm interfiber distance TD NIRS measurements. Time-point-spread-functions in 29- and 44-weeks postmenstrual age head meshes were simulated by the Monte Carlo method, convoluted with a real instrument response function, and then fitted with photon diffusion models. The results show good accuracy in retrieving brain oxygen saturation, and severe underestimation of total cerebral hemoglobin, suggesting the need for more complex models of analysis or of larger interfiber distances to precisely monitor all hemodynamic parameters.

On Synthetic Instrument Response Functions of Time-Correlated Single-Photon Counting Based Fluorescence Lifetime Imaging Analysis

Time-correlated single-photon counting (TCSPC) has been the gold standard for fluorescence lifetime imaging (FLIM) techniques due to its high signal-to-noize ratio and high temporal resolution. The sensor system's temporal instrument response function (IRF) should be considered in the deconvolution procedure to extract the real fluorescence decay to compensate for the distortion on measured decays contributed by the system imperfections. However, to measure the instrument response function is not trivial, and the measurement setup is different from measuring the real fluorescence. On the other hand, automatic synthetic IRFs can be directly derived from the recorded decay profiles and provide appropriate accuracy. This paper proposed and examined a synthetic IRF strategy. Compared with traditional automatic synthetic IRFs, the new proposed automatic synthetic IRF shows a broader dynamic range and better accuracy. To evaluate its performance, we examined simulated data using nonlinear least square deconvolution based on both the Levenberg-Marquardt algorithm and the Laguerre expansion method for bi-exponential fluorescence decays. Furthermore, experimental FLIM data of cells were also analyzed using the proposed synthetic IRF. The results from both the simulated data and experimental FLIM data show that the proposed synthetic IRF has a better performance compared to traditional synthetic IRFs. Our work provides a faster and precise method to obtain IRF, which may find various FLIM-based applications. We also reported in which conditions a measured or a synthesized IRF can be applied.

Continuous and discrete phasor analysis of binned or time-gated periodic decays.

The time-resolved analysis of periodically excited luminescence decays by the phasor method in the presence of time-gating or binning is revisited. Analytical expressions for discrete configurations of square gates are derived, and the locus of the phasors of such modified periodic single-exponential decays is compared to the canonical universal semicircle. The effects of instrument response function offset, decay truncation, and gate shape are also discussed. Finally, modified expressions for the phase and modulus lifetimes are provided for some simple cases. A discussion of a modified phasor calibration approach is presented, and an illustration of the new concepts with examples from the literature concludes this work.

Calibration of a CubeSat spectroradiometer with a narrow-band widely tunable radiance source.

We have developed an SI-traceable narrow-band tunable radiance source based on an optical parametric oscillator (OPO) and an integrating sphere for the calibration of spectroradiometers. The source is calibrated with a reference detector over the ultraviolet/visible spectral range with an uncertainty of <1%. As a case study, a CubeSat spectroradiometer has been calibrated for radiance over its operating range from 370 nm to 480 nm. To validate the results, the instrument has also been calibrated with a traditional setup based on a diffuser and an FEL lamp. Both routes show good agreement within the combined measurement uncertainty. The OPO-based approach could be an interesting alternative to the traditional method, not only because of reduced measurement uncertainty, but also because it directly allows for wavelength calibration and characterization of the instrumental spectral response function and stray light effects, which could reduce calibration time and cost.