Time-correlated Single Photon Counting Research Articles

Morphology-transforming AuNPs-based fluorescent probe for ultra-low sensitive detection of levofloxacin in urine samples at pH 7.0 through excimer formation

Fluorescent metal nanoparticles (MNPs) have garnered considerable interest in the realm of healthcare applications. Within this domain, we present a novel fluorescent sensor: Pamoic acid (PA) functionalized excitation-dependent gold nanoparticles (PA@AuNPs), designed to ascertain levofloxacin (LF) concentrations in human urine samples under pH 7.0 conditions. The synthesis of PA@AuNPs was achieved via a chemical reduction method employing PA as both a reducing and stabilizing agent. Validation of the successful synthesis of PA@AuNPs was conducted utilizing a suite of analytical techniques including X-ray diffraction (XRD), ultraviolet–visible absorption spectroscopy (UV–Vis), Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, and X-ray photoelectron spectroscopy (XPS). Microscopic examination via field emission scanning electron microscopy (FESEM) and high-resolution transmission electron microscopy (HRTEM) elucidated the spherical morphology of PA@AuNPs, with an average diameter ranging from 10 to 15 nm. Furthermore, atomic force microscopy (AFM) revealed an average surface roughness of 20.72 nm for PA@AuNPs. Thermal analysis results divulge that PA@AuNPs are highly stable up to 370 °C. The PA@AuNPs have shown more than 10-fold higher selectivity for LF in comparison with other commercially available antibiotics. The fluorescence emission intensity of PA@AuNPs (quantum yield (ΦF) 3.36 %) has shown enhanced redshift (436 → 497 nm; ∼61 nm) (λex 353 nm) upon the sequential addition of LF (0 → 100 µM) and achieved a lower detection limit (LoD = 36 nM; LoQ = 121 nM; Ka = 1.0457 × 104 M−1) with increased ΦF 18.96 %. This radiative process and fluorescence enhancement were confirmed by time-correlated single photon counting (TCSPC) measurement (4.89 → 5.36 ns). Interestingly, upon sensing LF, PA@AuNPs changed their morphology from spherical to octahedron and displayed an excimer formation through intermolecular π-π stacking. Furthermore, the stability of the complex, PA@AuNPs•LF which formed through coordination, by restoring the fluorescence intensity of PA@AuNPs•LF (turn on) in the presence of ethylenediamine tetraacetic acid (EDTA) (turn off) is demonstrated. The formation of the PA@AuNPs•LF complex was further confirmed by XRD analysis and FTIR spectroscopy. The HRTEM and FESEM validate that PA@AuNPs•LF have octahedral morphology with an average size of 20–30 nm, and the obtained moiré fringes in TEM images are due to the overlap of LF on the surface of PA@AuNPs. The AFM analysis divulges that PA@AuNPs•LF had a larger surface roughness (Sa = 26.91 nm). The experimentally attained zeta potential of PA@AuNPs (–12.6 mV) was further decreased (–17.6 mV) in the presence of LF, which confirms the interaction between PA@AuNPs and LF. The cell viability (82 %) in L929 Fibroblast cells augmented the biological applications of PA@AuNPs. The practicability of the fluorescent sensor probe was demonstrated in human urine samples with recovery ranges from 97.56 to 104.38 %.

Multimodal Multiphoton Tomography with a Compact Femtosecond Fiber Laser

Multiphoton tomography (MPT) based on near infrared (NIR) femtosecond laser technology has become a versatile high-resolution clinical and research imaging tool. We report on multimodal MPT with the air-cooled fiber laser tomograph MPTcompact. The ultracompact passively mode-locked erbium-doped 50/80 MHz laser operating at 780 nm is directly integrated into the 360° imaging head mounted on a flexible mechanical arm. The tomograph provides optical biopsies with subcellular resolution and optical metabolic imaging capability based on two-photon autofluorescence (AF), second harmonic generation, fluorescence lifetime imaging (FLIM) by time-correlated single photon counting with 250 ps temporal resolution, reflectance confocal microscopy, and white LED light CMOS camera imaging. For the first time, NIR femtosecond laser pulses have been used on human skin to realize simultaneous one-photon confocal imaging and two-photon imaging. Most useful information is provided by the two-photon excited intratissue AF of the coenzymes NAD(P)H and flavins. The signals of both types of coenzymes can be separated by FLIM. Furthermore, the free and protein-bound forms can be distinguished by time-resolved AF detection because the protein-bound NADH has one order higher AF lifetime than free (non-bound) NADH. The tomograph contains onboard storage batteries so that it can operate for up to a few hours independently from external power supply. Applications include in vivo cancer detection and in situ evaluation of anti-aging drugs and pharmaceuticals. The tomograph MPTcompact has been successfully tested in a clinical multicenter study for the diagnosis of malignant melanoma on 100 patients with suspicious pigmented lesions. MPT has the potential to realize non-invasive high-resolution label-free in vivo histology within minutes and, therefore, to reduce the number of physically taken biopsies.

Photon counting based pump-probe technique for quantitative characterization of fluorescence in a lock-in free detection manner

The stimulated emission (SE) signal in pump-probe experiment is conventionally measured with lock-in detection to differentiate the weak signals from the relatively large background of spontaneous emission and probe beam. Therefore, direct characterization of signal strength are often major limiting factors in terms of noise, speed, and data acquisition. In contrast, photon counting allows direct quantification of signal strength, while synchronized pump-probe pulse enables precise timing and the separation of signals accordingly. Herein, the SE based pump-probe method is combined with time-correlated single-photon counting to investigate the ultrafast photochemical parameters, digitally and quantitatively. As a proof-of-concept, our technique is applied to investigate, fluorescence lifetime (τ)∼3.71ns , optical absorption cross-section (σabs)∼1.23×10−16cm2 , and the SE cross-section (σSE)∼2.22×10−17cm2 , of a fluorescent dye (ATTO 647N) quantitatively. The experimental results are also compared with theoretical photon statistics to further justify the advantages including experimental and statistical critical molecular dynamics parameters extraction with excellent high accuracy.

2D signal estimation for sparse distributed target photon counting data

In this study, we explore the utilization of penalized likelihood estimation for the analysis of sparse photon counting data obtained from distributed target lidar systems. Specifically, we adapt the Poisson Total Variation processing technique to cater to this application. By assuming a Poisson noise model for the photon count observations, our approach yields denoised estimates of backscatter photon flux and related parameters. This facilitates the processing of raw photon counting signals with exceptionally high temporal and range resolutions (demonstrated here to 50 Hz and 75 cm resolutions), including data acquired through time-correlated single photon counting, without significant sacrifice of resolution. Through examination involving both simulated and real-world 2D atmospheric data, our method consistently demonstrates superior accuracy in signal recovery compared to the conventional histogram-based approach commonly employed in distributed target lidar applications.

Bismuth (Bi3+) based lead-free halide perovskitoid for light driven applications: A potential low-toxic alternative for lead (Pb2+)

Bismuth-based perovskite-like materials are recognised as feasible optoelectronic alternatives to lead-based perovskites. High toxicity and limited environmental stability of Pb-based perovskite are the principal hurdles for their practical use, whereas Bismuth (Bi3+) based materials exhibit relatively low toxicity. In this study, we fabricated (FPEA)BiI4 (FPEA = Fluorophenylethyl amine, as a hydrophobic aromatic spacer) and explored its compatibility in the field of photocatalysis and solar cells. UV–Vis, photoluminescence (PL) and time-correlated single photon counting (TCSPC) techniques were used to understand the optoelectronic properties of the material. These fabricated thin films were used towards toxic MBT remediation. Solar cells (in n-i-p configuration) were also fabricated using (FPEA)BiI4 and their photo conversion efficiency (PCE) was tested under AM1.5 illumination.

Fluctuations in the Photoluminescence Excitation Spectra of Individual Semiconductor Nanocrystals.

Most single quantum emitters display non-steady emission properties. Models that explain this effect have primarily relied on photoluminescence measurements that reveal variations in intensity, wavelength, and excited-state lifetime. While photoluminescence excitation spectroscopy could provide complementary information, existing experimental methods cannot collect spectra before individual emitters change in intensity (blink) or wavelength (spectrally diffuse). Here, we present an experimental approach that circumvents such issues, allowing the collection of excitation spectra from individual emitters. Using rapid modulation of the excitation wavelength, we collect and classify excitation spectra from individual CdSe/CdS/ZnS core/shell/shell quantum dots. The spectra, along with simultaneous time-correlated single-photon counting, reveal two separate emission-reduction mechanisms caused by charging and trapping, respectively. During bright emission periods, we also observe a correlation between emission red-shifts and the increased oscillator strength of higher excited states. Quantum-mechanical modeling indicates that diffusion of charges in the vicinity of an emitter polarizes the exciton and transfers the oscillator strength to higher-energy transitions.

Photon counting Raman spectroscopy: a benchmarking study vs surface plasmon enhancement

We demonstrate a single-photon counting Raman spectroscope and benchmark it against conventional and surface-enhanced Raman spectroscopy. For direct comparison without ambiguity, we use the same solutions of Rhodamine 6G and a common optical setup with either a spectrometer or an acousto-optic tunable filter, whereas the surface enhancement is realized with immobilized Ag nanoparticles. Our results find that the single photon counting significantly elevates the detection sensitivity by up to eight orders of magnitude, arriving at a comparable level of surface-enhanced Raman spectroscopy. Another significant advantage is with the time-resolving measurement, where we demonstrate time-gated and time-correlated single-photon counting with sub-nanosecond resolution. It offers insights into the samples’ transient responses and enables the isolation of Raman scattering from fluorescence signals.

A single-photon lidar observes atmospheric clouds at decimeter scales: resolving droplet activation within cloud base

Clouds, crucial for understanding climate, begin with droplet formation from aerosols, but observations of this fleeting activation step are lacking in the atmosphere. Here we use a time-gated time-correlated single-photon counting lidar to observe cloud base structures at decimeter scales. Results show that the air–cloud interface is not a perfect boundary but rather a transition zone where the transformation of aerosol particles into cloud droplets occurs. The observed distributions of first-arriving photons within the transition zone reflect vertical development of a cloud, including droplet activation and condensational growth. Further, the highly resolved vertical profile of backscattered photons above the cloud base enables remote estimation of droplet concentration, an elusive but critical property to understanding aerosol–cloud interactions. Our results show the feasibility of remotely monitoring cloud properties at submeter scales, thus providing much-needed insights into the impacts of atmospheric pollution on clouds and aerosol-cloud interactions that influence climate.

Intra- and Intermolecular Charge-Transfer Dynamics of Carbene-Metal-Amide Photosensitizers.

A series of steady-state and time-resolved spectroscopies were performed on a set of eight carbene-metal-amide (cMa) complexes, where M = Cu and Au, that have been used as photosensitizers for photosensitized electrocatalytic reactions. Using ps-to-ns and ns-to-ms transient absorption spectroscopies (psTA and nsTA, respectively), the excited-state kinetics from light absorption, intersystem crossing (ISC), and eventually intermolecular charge transfer were thoroughly characterized. Using time-correlated single photon counting (TCSPC) and psTA with a thermally activated delayed fluorescence (TADF) model, the variation in intersystem crossing (ISC), (S1 → T1) rates (∼3-120 × 109 s-1), and ΔEST values (73-115 meV) for these compounds were fully characterized, reflecting systematic changes to the carbene, carbazole, and metal. The psTA additionally revealed an early time relaxation (rate ∼0.2-0.8 × 1012 s-1) attributed to solvent relaxation and vibrational cooling. The nsTA experiments for a gold-based cMa complex demonstrated efficient intermolecular charge transfer from the excited cMa to an electron acceptor. Pulse radiolysis and bulk electrolysis experiments allowed us to identify the character of the transient excited states as ligand-ligand charge transfer as well as the spectroscopic signature of oxidized and reduced forms of the cMa photosensitizer.

Cellular Imaging and Time-Domain FLIM Studies of Meso-Tetraphenylporphine Disulfonate as a Photosensitising Agent in 2D and 3D Models.

Fluorescence lifetime imaging (FLIM) and confocal fluorescence studies of a porphyrin-based photosensitiser (meso-tetraphenylporphine disulfonate: TPPS2a) were evaluated in 2D monolayer cultures and 3D compressed collagen constructs of a human ovarian cancer cell line (HEY). TPPS2a is known to be an effective model photosensitiser for both Photodynamic Therapy (PDT) and Photochemical Internalisation (PCI). This microspectrofluorimetric study aimed firstly to investigate the uptake and subcellular localisation of TPPS2a, and evaluate the photo-oxidative mechanism using reactive oxygen species (ROS) and lipid peroxidation probes combined with appropriate ROS scavengers. Light-induced intracellular redistribution of TPPS2a was observed, consistent with rupture of endolysosomes where the porphyrin localises. Using the same range of light doses, time-lapse confocal imaging permitted observation of PDT-induced generation of ROS in both 2D and 3D cancer models using fluorescence-based ROS together with specific ROS inhibitors. In addition, the use of red light excitation of the photosensitiser to minimise auto-oxidation of the probes was investigated. In the second part of the study, the photophysical properties of TPPS2a in cells were studied using a time-domain FLIM system with time-correlated single photon counting detection. Owing to the high sensitivity and spatial resolution of this system, we acquired FLIM images that enabled the fluorescence lifetime determination of the porphyrin within the endolysosomal vesicles. Changes in the lifetime dynamics upon prolonged illumination were revealed as the vesicles degraded within the cells.

Orange-Red-Emitting Carbon Dots for Bilirubin Detection and Its Antibacterial Activity Against Escherichia coli and Staphylococcus aureus.

In this study, orange-red-emitting carbon dots (OR-CDs) were prepared from p-phenylenediamine (p-PDA) and urea as starting precursors through the hydrothermal method. The OR-CDs exhibited bright orange-red fluorescence at 618 nm when excited at 480 nm. The obtained OR-CDs exhibited stable photophysical properties under different physiological conditions. The unique photophysical property of OR-CDs were then utilized for fluorometric determination of bilirubin. The fluorometric assay revealed that the fluorescence intensity of OR-CDs is gradually quenched upon the addition of bilirubin (1-20 μM). The mechanism of fluorescence quenching was evaluated by steady-state fluorescence analysis and time-correlated single photon counting measurements. The OR-CDs showed good selectivity and sensitivity toward bilirubin over other common interfering biomolecules. The present fluorometric assay showed a linear response toward bilirubin between 1 and 10 μM with a limit of detection of 4.80 nM. Further, a fluorescence test cotton swab-based detection probe has been successfully developed by incorporating OR-CDs for the point-of-care detection of bilirubin in biofluids. Furthermore, a light-emitting diode light that emits orange-red light was prepared by embedding the OR-CDs within the poly(vinyl alcohol) polymer matrix. Moreover, the antibacterial activity of OR-CDs was tested against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. The antibacterial efficacy of OR-CDs was demonstrated by various mechanisms, such as reactive oxygen species generation, destruction of cell structure, chemical binding to membrane, and surface wrapping. Interestingly, the survival assay against L929 fibroblast cells exhibits favorable biocompatibility and bioimaging.

Optical and structural properties of chlorophyll a aggregates in PVA film

The aggregated properties of Chlorophyll a (Chla) in polymer play a pivotal role in light capture and transfer as photostable pigment sensitizers of solar power systems. Here, we embedded the natural Chla molecules (2 × 10−3 M) extracted from spinach leaves into polyvinyl alcohol (PVA) films by casting method under different temperature. UV–VIS, fluorescence, Circular Dichroism (CD) spectroscopy, and Transmission Electron Microscopy (SEM) have been applied to investigate the optical properties and aggregated structures in detail. The excited state dynamics of these aggregates in PVA were explored using Time-Correlated Single Photon Counting (TCSPC). The de-composited components from absorption, emission, TCSPC and the CD shapes in the study show the analogous characteristics to the photosystem of cyanobacteria in some papers that features a coexisting of several aggregates (dimer, oligomer, bulk oligomer), in which the proportion of bulk oligomer can be greatly promoted under lower temperature (0 °C) due to the involvement of a large amount of water. The analysis of spectral and dynamical components reveals the bulk oligomer model of Chla in PVA, which is constructed from several oligomers by PVA chain and water. Besides, a specific red trapping center within bulk oligomer is proposed to perform capturing energy in 18 ps, which is located on the lowest end of energy flow and devotes large than 99% florescence in TCSPC.

A time-correlated single photon counting SPAD array camera with a bespoke data-processing algorithm for lightsheet fluorescence lifetime imaging (FLIM) and FLIM videos

A wide-field microscope with epi-fluorescence and selective plane illumination was combined with a single-photon avalanche diode (SPAD) array camera to enable live-cell fluorescence lifetime imaging (FLIM) using time-correlated single-photon counting (TCSPC). The camera sensor comprised of 192×128\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\ ext {192}\ imes \ ext {128}$$\\end{document} pixels, each integrating a single SPAD and a time-to-digital converter. Jointly, they produced a stream of single-photon images of photon arrival times with ≈38 ps\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\approx \ ext {38 ps}$$\\end{document} accuracy. The photon arrival times were subject to systematic delays and nonlinearities, which were corrected by a Monte-Carlo algorithm. The SPAD camera was then applied to FLIM where histogramming the resulting photon arrival times in each pixel resulted in decays compatible with common data processing pipelines for fluorescence lifetime analysis. The capabilities of the TCSPC camera-based FLIM microscope were demonstrated by imaging living unicellular photosynthetic algae and artificial lipid vesicles. Epi-fluorescence illumination enabled rapid fluorescence lifetime imaging of living cells and selective-plane illumination enabled 3-dimensional FLIM of stationary samples.

Blind instrument response function identification from fluorescence decays

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

Enhancing the spatial resolution of time-of-flight based non-line-of-sight imaging via instrument response function deconvolution.

Non-line-of-sight (NLOS) imaging retrieves the hidden scenes by utilizing the signals indirectly reflected by the relay wall. Benefiting from the picosecond-level timing accuracy, time-correlated single photon counting (TCSPC) based NLOS imaging can achieve theoretical spatial resolutions up to millimeter level. However, in practical applications, the total temporal resolution (also known as total time jitter, TTJ) of most current TCSPC systems exceeds hundreds of picoseconds due to the combined effects of multiple electronic devices, which restricts the underlying spatial resolution of NLOS imaging. In this paper, an instrument response function deconvolution (IRF-DC) method is proposed to overcome the constraints of a TCSPC system's TTJ on the spatial resolution of NLOS imaging. Specifically, we model the transient measurements as Poisson convolution process with the normalized IRF as convolution kernel, and solve the inverse problem with iterative deconvolution algorithm, which significantly improves the spatial resolution of NLOS imaging after reconstruction. Numerical simulations show that the IRF-DC facilitates light-cone transform and frequency-wavenumber migration solver to achieve successful reconstruction even when the system's TTJ reaches 1200 ps, which is equivalent to what was previously possible when TTJ was about 200ps. In addition, the IRF-DC produces satisfactory reconstruction outcomes when the signal-to-noise ratio (SNR) is low. Furthermore, the effectiveness of the proposed method has also been experimentally verified. The proposed IRF-DC method is highly applicable and efficient, which may promote the development of high-resolution NLOS imaging.

Quantifying distortion in time-correlated single photon counting: a universal parameter.

One major drawback of the classic time-correlated single photon counting (TCSPC) technique is pileup-related distortion. To keep it under a reasonable level, the maximum count rate has to be reduced, posing a serious limitation to the overall measurement speed. This means that there is an intrinsic trade-off between speed and distortion: either count rate is raised, but distortion is worsened, or distortion is minimized at the expense of speed. In both cases, it is impossible to precisely evaluate the degree of distortion introduced. Here comes our new, to the best of our knowledge, figure of merit, which is able to provide a numerical estimate of the distortion whatever the signal shape is, marking a real turning point in the way of doing TCSPC. In this article, this new parameter will be defined and its effectiveness will be demonstrated by means of mathematical computations.

Miniaturized time-correlated single-photon counting module for time-of-flight non-line-of-sight imaging applications.

Single-photon time-of-flight (TOF) non-line-of-sight (NLOS) imaging enables the high-resolution reconstruction of objects outside the field of view. The compactness of TOF NLOS imaging systems, entailing the miniaturization of key components within such systems, is crucial for practical applications. Here, we present a miniaturized four-channel time-correlated single-photon counting module dedicated to TOF NLOS imaging applications. The module achieves excellent performance with a 10ps bin size and 27.4ps minimum root-mean-square time resolution. We present the results of the TOF NLOS imaging experiment using an InGaAs/InP single-photon detector and the time-correlated single-photon counting module and show that a 6.3cm lateral resolution and 2.3cm depth resolution can be achieved under the conditions of 5m imaging distance and 1ms pixel dwell time.

Ultrafast energy transfer dynamics in CsPbBr3 nanoplatelets-BODIPY heterostructure.

Understanding and directing the energy transfer in nanocrystals-chromophore heterostructure is critical to improve the efficiency of their photocatalytic and optoelectronic applications. In this work, we studied the energy transfer process between inorganic-organic molecular complexes composed of cesium halide perovskite nanoplatelets (CsPbBr3 NPLs) and boron dipyrromethene (BODIPY) by photoluminescence spectroscopy (PL), time-correlated single photon-counting (TCSPC) and femtosecond transient absorption spectroscopy. The quenching of PL in CsPbBr3 NPLs occurred simultaneously with the PL enhancement of BODIPY implied the singlet energy transfer process. The rate of energy transfer has been determined by transient absorption spectrum as kET = 3.8 × 109 s-1. The efficiency of Förster energy transfer (FRET) has been quantitatively calculated up to 70%. Our work advances the understanding of the interaction between BODIPY and perovskite nanoplatelets, providing a new solution based on their optoelectronic and photocatalytic applications.

Chiral-Induced Spin Selectivity Modulated Time-Correlated Single-Photon Counting for DNA Hybridization Detection.

The chiral-induced spin selectivity (CISS) effect can distinguish between the spin of electrons as they pass through chiral molecules by backscattering one of the spin components. Herein, we explore the role of the CISS effect in time-correlated single-photon counting measurements to detect DNA hybridization. We observe that the average lifetime of optical excited states of quantum dots attached to double-stranded DNA (dsDNA) varies with directions of the applied magnetic field. Specifically, the difference in the nonradiative average decay lifetime for the two orientations of the applied magnetic field is 2.21 ns in the case of hybridized strands, which is 130 times higher than that observed with quantum dots attached to single-strand DNA. Additionally, we investigate the application of Fourier transform infrared (FTIR) spectroscopy for detecting double-stranded DNA in the presence of a magnetic field, establishing a theoretical framework to substantiate the experimental evidence of magnetic field-dependent FTIR spectroscopy for dsDNA.

Wide-field illumination diffuse optical tomography within a framework of single-pixel time-domain spatial frequency domain imaging.

We present a wide-field illumination time-domain (TD) diffusion optical tomography (DOT) for three-dimensional (3-D) reconstruction within a shallow region under the illuminated surface of the turbid medium. The methodological foundation is laid on the single-pixel spatial frequency domain (SFD) imaging that facilitates the adoption of the well-established time-correlated single-photon counting (TCSPC)-based TD detection and generalized pulse spectrum techniques (GPST)-based reconstruction. To ameliorate the defects of the conventional diffusion equation (DE) in the forward modeling of TD-SFD-DOT, mainly the low accuracy in the near-field region and in profiling early-photon migration, we propose a modified model employing the time-dependent δ-P1 approximation and verify its improved accuracy in comparison with both the Monte Carlo and DE-based ones. For a simplified inversion process, a modified GPST approach is extended to TD-SFD-DOT that enables the effective separation of the absorption and scattering coefficients using a steady-state equivalent strategy. Furthermore, we set up a single-pixel TD-SFD-DOT system that employs the TCSPC-based TD detection in the SFD imaging framework. For assessments of the reconstruction approach and the system performance, phantom experiments are performed for a series of scenarios. The results show the effectiveness of the proposed methodology for rapid 3-D reconstruction of the absorption and scattering coefficients within a depth range of about 5 mean free pathlengths.