Hard X-ray full-field nanoimaging using a direct photon-counting detector.

Full-field X-ray nanoimaging is a widely used tool in a broad range of scientific areas. In particular, for low-absorbing biological or medical samples, phase contrast methods have to be considered. Three well established phase contrast methods at the nanoscale are transmission X-ray microscopy with Zernike phase contrast, near-field holography and near-field ptychography. The high spatial resolution, however, often comes with the drawback of a lower signal-to-noise ratio and significantly longer scan times, compared with microimaging. In order to tackle these challenges a single-photon-counting detector has been implemented at the nanoimaging endstation of the beamline P05 at PETRA III (DESY, Hamburg) operated by Helmholtz-Zentrum Hereon. Thanks to the long sample-to-detector distance available, spatial resolutions of below 100 nm were reached in all three presented nanoimaging techniques. This work shows that a single-photon-counting detector in combination with a long sample-to-detector distance allows one to increase the time resolution for in situ nanoimaging, while keeping a high signal-to-noise level.

Nanoscale imaging of quantum dot dimers using time-resolved super-resolution microscopy combined with scanning electron microscopy.

Time-resolved super-resolution microscopy was used in conjunction with scanning electron microscopy to image individual colloidal CdSe/CdS semiconductor quantum dots (QD) and QD dimers. The photoluminescence (PL) lifetimes, intensities, and structural parameters were acquired with nanometer scale spatial resolution and sub-nanosecond time resolution. The combination of these two techniques was more powerful than either alone, enabling us to resolve the PL properties of individual QDs within QD dimers as they blinked on and off, measure interparticle distances, and identify QDs that may be participating in energy transfer. The localization precision of our optical imaging technique was ∼3 nm, low enough that the emission from individual QDs within the dimers could be spatially resolved. While the majority of QDs within dimers acted as independent emitters, at least one pair of QDs in our study exhibited lifetime and intensity behaviors consistent with resonance energy transfer from a shorter lifetime and lower intensity donor QD to a longer lifetime and higher intensity acceptor QD. For this case, we demonstrate how the combined super-resolution optical imaging and scanning electron microscopy data can be used to characterize the energy transfer rate.

Time-resolved emission spectroscopy to elucidate the functional nature of heat-stable transcription factor.

A 22 kDa protein from Thermus thermophilus is characterised as a DNA binding transcription regulator and its function is established using the fluorescence spectroscopy technique. The steady-state fluorescence spectroscopy result shows significant binding of calf thymus DNA and protein molecule. To confirm, the DNA quenching effect in real-time, a time-resolved emission spectroscopy study was performed and the result shows good agreement with steady-state quenching analysis.

Free serum sorbitol and its interaction with caffeine: A suggestive approach for plausible remediation of diabetic neuropathy.

The measure of sorbitol in serum can act as a good indicator in the monitoring of the diabetic complications. To analyze the sorbitol level in serum medium, fluorometric enzymatic assay was performed. To remove the excess sorbitol from the body, proposed binding of sorbitol with caffeine was investigated. Their interaction in serum medium was studied and established by UV-Vis, fluorescence spectrophotometry, and time-correlated single photon counting (TCSPC). The linear calibration of sorbitol (in the range 10-50 mM) was done using UV-Vis spectrophotometry. Time scan experiments furnished the reaction rate of sorbitol assayed solution as well as sorbitol-caffeine complex as 0.021 min-1 and 0.018 min-1 , respectively. A sudden drop was observed in the fluorescence lifetime of reduced nicotinamide adenine dinucleotide (NADH) present in sorbitol assayed solution upon complexation with caffeine, that is, from 1.774 × 10-09 to 1.23 × 10-10 Sec, which indicates the hindrance in the formation of NADH and the probable formation of some other species. Isothermal titration calorimetric experiments clearly indicate the number of binding sites (i.e., 3.89, 1.40, and 2.07) that exist between sorbitol and caffeine at the complexation ratio of 1:1.2, 1:1.5, and 1:3. The present method can be helpful in pharmacological and therapeutic studies of sorbitol using caffeine for treating diabetic neuropathy.

Characterization and performance evaluation of the XSPA-500k detector using synchrotron radiation.

Hybrid photon counting (HPC) detectors are widely used at both synchrotron facilities and in-house laboratories. The features of HPC detectors, such as no readout noise, high dynamic range, high frame rate, excellent point spread function, no blurring etc. along with fast data acquisition, provide a high-performance detector with a low detection limit and high sensitivity. Several HPC detector systems have been developed around the world. A number of them are commercially available and used in academia and industry. One of the important features of an HPC detector is a fast readout speed. Most HPC detectors can easily achieve over 1000 frames s-1, one or two orders of magnitude faster than conventional CCD detectors. Nevertheless, advanced scientific challenges require ever faster detectors in order to study dynamical phenomena in matter. The XSPA-500k detector can achieve 56 kframes s-1 continuously, without dead-time between frames. Using `burst mode', a special mode of the UFXC32k ASIC, the frame rate reaches 1 000 000 frames s-1. XSPA-500k was fully evaluated at the Metrology beamline at Synchrotron SOLEIL (France) and its readout speed was confirmed by tracking the synchrotron bunch time structure. The uniformity of response, modulation transfer function, linearity, energy resolution and other performance metrics were also verified either with fluorescence X-rays illuminating the full area of the detector or with the direct beam.

Single Photon Avalanche Diode Arrays for Time-Resolved Raman Spectroscopy.

The detection of peaks shifts in Raman spectroscopy enables a fingerprint reconstruction to discriminate among molecules with neither labelling nor sample preparation. Time-resolved Raman spectroscopy is an effective technique to reject the strong fluorescence background that profits from the time scale difference in the two responses: Raman photons are scattered almost instantaneously while fluorescence shows a nanoseconds time constant decay. The combination of short laser pulses with time-gated detectors enables the collection of only those photons synchronous with the pulse, thus rejecting fluorescent ones. This review addresses time-gating issues from the sensor standpoint and identifies single photon avalanche diode (SPAD) arrays as the most suitable single-photon detectors to be rapidly and precisely time-gated without bulky, complex, or expensive setups. At first, we discuss the requirements for ideal Raman SPAD arrays, particularly focusing on the design guidelines for optimized on-chip processing electronics. Then we present some existing SPAD-based architectures, featuring specific operation modes which can be usefully exploited for Raman spectroscopy. Finally, we highlight key aspects for future ultrafast Raman platforms and highly integrated sensors capable of undistorted identification of Raman peaks across many pixels.

Comparison of Time Resolved Optical Turbidity Measurements for Water Monitoring to Standard Real-Time Techniques.

Environmental water monitoring requires the estimation of the suspended solids load. In this paper, we compare the concentration range accessible through three different techniques: optical turbidity, acoustic backscattering and the newly in-lab developed time resolved optical turbidity. We focus on their comparison on measurements made in the laboratory on water suspensions of known particles and concentrations. We used laboratory grade kieselguhr, wheat starch and kaolin as suspended solid surrogates. The explored concentration domains are the ones, for the total suspended solid load, commonly encountered in wastewater and rivers in standard (less than 1 g/L to a few g/L) or extreme conditions such as floods or storm events (up to several dozen g/L). Regarding the operable concentration domain, the time resolved optical turbidity shows a clear advantage upon the other methods, whatever the kind of particle is.

Ultrafast Single-Molecule Fluorescence Measured by Femtosecond Double-Pulse Excitation Photon Antibunching.

Most measurements of fluorescence lifetimes on the single-molecule level are carried out using avalanche photon diodes (APDs). These single-photon counters are inherently slow, and their response shows a strong dependence on photon energy, which can make reconvolution of the instrument response function (IRF) challenging. An ultrafast time resolution in single-molecule fluorescence is crucial, e.g., in determining donor lifetimes in donor-acceptor couples which undergo energy transfer, or in plasmonic antenna structures, where the radiative rate and non-radiative rates are enhanced. We introduce a femtosecond double-excitation (FeDEx) photon correlation technique, which measures the degree of photon antibunching as a function of time delay between two excitation pulses. In this boxcar integration, the time resolution of fluorescence transients is limited solely by the laser pulse length and is independent of the detector IRF. The versatility of the technique is demonstrated with a custom-made donor-acceptor complex with one donor and two acceptors and with single dye molecules positioned accurately between two gold nanoparticles using DNA origami. The latter structures show ∼75-fold radiative-rate enhancement and fluorescence lifetimes down to 19 ps, which is measured without the need for any reconvolution. With the potential of measuring subpicosecond fluorescence lifetimes, plasmonic antenna structures can now be optimized further.

Time-Resolved Fluorescence Anisotropy of a Molecular Rotor Resolves Microscopic Viscosity Parameters in Complex Environments.

Understanding viscosity in complex environments remains a largely unanswered question despite its importance in determining reaction rates in vivo. Here, time-resolved fluorescence anisotropy imaging (TR-FAIM) is combined with fluorescent molecular rotors (FMRs) to simultaneously determine two non-equivalent viscosity-related parameters in complex heterogeneous environments. The parameters, FMR rotational correlation time and lifetime, are extracted from fluorescence anisotropy decays, which in heterogeneous environments show dip-and-rise behavior due to multiple dye populations. Decays of this kind are found both in artificially constructed adiposomes and in live cell lipid droplet organelles. Molecular dynamics simulations are used to assign each population to nano-environments within the lipid systems. The less viscous population corresponds to the state showing an average 25° tilt to the lipid membrane normal, and the more viscous population to the state showing an average 55° tilt. This combined experimental and simulation approach enables a comprehensive description of the FMR probe behavior within viscous nano-environments in complex, biological systems.

Real-Time Dual-Wavelength Time-Resolved Diffuse Optical Tomography System for Functional Brain Imaging Based on Probe-Hosted Silicon Photomultipliers.

Near-infrared diffuse optical tomography is a non-invasive photonics-based imaging technology suited to functional brain imaging applications. Recent developments have proved that it is possible to build a compact time-domain diffuse optical tomography system based on silicon photomultipliers (SiPM) detectors. The system presented in this paper was equipped with the same eight SiPM probe-hosted detectors, but was upgraded with six injection fibers to shine the sample at several points. Moreover, an automatic switch was included enabling a complete measurement to be performed in less than one second. Further, the system was provided with a dual-wavelength ( 670 n m and 820 n m ) light source to quantify the oxy- and deoxy-hemoglobin concentration evolution in the tissue. This novel system was challenged against a solid phantom experiment, and two in-vivo tests, namely arm occlusion and motor cortex brain activation. The results show that the tomographic system makes it possible to follow the evolution of brain activation over time with a 1 s -resolution.

Counting of Hong-Ou-Mandel Bunched Optical Photons Using a Fast Pixel Camera.

The uses of a silicon-pixel camera with very good time resolution (∼nanosecond) for detecting multiple, bunched optical photons is explored. We present characteristics of the camera and describe experiments proving its counting capabilities. We use a spontaneous parametric down-conversion source to generate correlated photon pairs, and exploit the Hong-Ou-Mandel (HOM) interference effect in a fiber-coupled beam splitter to bunch the pair onto the same output fiber. It is shown that the time and spatial resolution of the camera enables independent detection of two photons emerging simultaneously from a single spatial mode.

Fluorescence Lifetime and Intensity of Thioflavin T as Reporters of Different Fibrillation Stages: Insights Obtained from Fluorescence Up-Conversion and Particle Size Distribution Measurements.

Thioflavin T (ThT) assay is extensively used for studying fibrillation kinetics in vitro. However, the differences in the time course of ThT fluorescence intensity and lifetime and other physical parameters of the system, such as particle size distribution, raise questions about the correct interpretation of the aggregation kinetics. In this work, we focused on the investigation of the mechanisms, which underlay the difference in sensitivity of ThT fluorescence intensity and lifetime to the formation of protein aggregates during fibrillation by the example of insulin and during binding to globular proteins. The assessment of aggregate sizes and heterogeneity was performed using dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). Using the sub-nanosecond resolution measurements, it was shown that the ThT lifetime is sensitive to the appearance of as much as a few percent of ThT bound to the high-affinity sites that occur simultaneously with an abrupt increase of the average particle size, particles concentration, and size heterogeneity. The discrepancy between ThT fluorescence intensity and a lifetime can be explained as the consequence of a ThT molecule fraction with ultrafast decay and weak fluorescence. These ThT molecules can only be detected using time-resolved fluorescence measurements in the sub-picosecond time domain. The presence of a bound ThT subpopulation with similar photophysical properties was also demonstrated for globular proteins that were attributed to non-specifically bound ThT molecules with a non-rigid microenvironment.

Development of low-energy X-ray detectors using LGAD sensors.

Recent advances in segmented low-gain avalanche detectors (LGADs) make them promising for the position-sensitive detection of low-energy X-ray photons thanks to their internal gain. LGAD microstrip sensors fabricated by Fondazione Bruno Kessler have been investigated using X-rays with both charge-integrating and single-photon-counting readout chips developed at the Paul Scherrer Institut. In this work it is shown that the charge multiplication occurring in the sensor allows the detection of X-rays with improved signal-to-noise ratio in comparison with standard silicon sensors. The application in the tender X-ray energy range is demonstrated by the detection of the sulfur Kα and Kß lines (2.3 and 2.46 keV) in an energy-dispersive fluorescence spectrometer at the Swiss Light Source. Although further improvements in the segmentation and in the quantum efficiency at low energy are still necessary, this work paves the way for the development of single-photon-counting detectors in the soft X-ray energy range.

Advancements towards the implementation of clinical phase-contrast breast computed tomography at Elettra.

Breast computed tomography (BCT) is an emerging application of X-ray tomography in radiological practice. A few clinical prototypes are under evaluation in hospitals and new systems are under development aiming at improving spatial and contrast resolution and reducing delivered dose. At the same time, synchrotron-radiation phase-contrast mammography has been demonstrated to offer substantial advantages when compared with conventional mammography. At Elettra, the Italian synchrotron radiation facility, a clinical program of phase-contrast BCT based on the free-space propagation approach is under development. In this paper, full-volume breast samples imaged with a beam energy of 32 keV delivering a mean glandular dose of 5 mGy are presented. The whole acquisition setup mimics a clinical study in order to evaluate its feasibility in terms of acquisition time and image quality. Acquisitions are performed using a high-resolution CdTe photon-counting detector and the projection data are processed via a phase-retrieval algorithm. Tomographic reconstructions are compared with conventional mammographic images acquired prior to surgery and with histologic examinations. Results indicate that BCT with monochromatic beam and free-space propagation phase-contrast imaging provide relevant three-dimensional insights of breast morphology at clinically acceptable doses and scan times.

Large-area single-photon-counting CdTe detector for synchrotron radiation computed tomography: a dedicated pre-processing procedure.

Large-area CdTe single-photon-counting detectors are becoming more and more attractive in view of low-dose imaging applications due to their high efficiency, low intrinsic noise and absence of a scintillating screen which affects spatial resolution. At present, however, since the dimensions of a single sensor are small (typically a few cm2), multi-module architectures are needed to obtain a large field of view. This requires coping with inter-module gaps and with close-to-edge pixels, which generally show a non-optimal behavior. Moreover, high-Z detectors often show gain variations in time due to charge trapping: this effect is detrimental especially in computed tomography (CT) applications where a single tomographic image requires hundreds of projections continuously acquired in several seconds. This work has been carried out at the SYRMEP beamline of the Elettra synchrotron radiation facility (Trieste, Italy), in the framework of the SYRMA-3D project, which aims to perform the world's first breast-CT clinical study with synchrotron radiation. An ad hoc data pre-processing procedure has been developed for the PIXIRAD-8 CdTe single-photon-counting detector, comprising an array of eight 30.7 mm × 24.8 mm modules tiling a 246 mm × 25 mm sensitive area, which covers the full synchrotron radiation beam. The procedure consists of five building blocks, namely dynamic flat-fielding, gap seaming, dynamic ring removal, projection despeckling and around-gap equalization. Each block is discussed and compared, when existing, with conventional approaches. The effectiveness of the pre-processing is demonstrated for phase-contrast CT images of a human breast specimen. The dynamic nature of the proposed procedure, which provides corrections dependent upon the projection index, allows the effective removal of time-dependent artifacts, preserving the main image features including phase effects.

Evaluation of the UFXC32k photon-counting detector for pump-probe experiments using synchrotron radiation.

This paper presents the performance of a single-photon-counting hybrid pixel X-ray detector with synchrotron radiation. The camera was evaluated with respect to time-resolved experiments, namely pump-probe-probe experiments held at SOLEIL. The UFXC camera shows very good energy resolution of around 1.5 keV and allows the minimum threshold setting to be as low as 3 keV keeping the high-count-rate capabilities. Measurements of a synchrotron characteristic filling mode prove the proper separation of an isolated bunch of photons and the usability of the detector in time-resolved experiments.

Red-Enhanced Photon Detection Module Featuring a 32 × 1 Single-Photon Avalanche Diode Array.

In this letter, the development and the experimental characterization of a new photon detection module, based on a 32×1 red-enhanced single-photon avalanche diode (RE-SPAD) array, are presented. A custom-developed technology has been exploited to design a detector having large-area pixels (50-µm diameter) with optimized performance. With an excess bias voltage Voυ = 15 V, a photon detection efficiency as high as 57% at 600 nm (33% at 800 nm) is achieved, along with dark count rate in the kHz range and optical crosstalk probability as low as 0.29%. The remarkable detection efficiency of the RE-SPAD array makes the module particularly suitable for all applications where high detection efficiency in the red/near-infrared range is mandatory. As an example, the performance of the array module is demonstrated to match the demanding requirements of multispot single-molecule fluorescence spectroscopy.

Lightweight Raman spectroscope using time-correlated photon-counting detection.

Raman spectroscopy is an important tool in understanding chemical components of various materials. However, the excessive weight and energy consumption of a conventional CCD-based Raman spectrometer forbids its applications under extreme conditions, including unmanned aircraft vehicles (UAVs) and Mars/Moon rovers. In this article, we present a highly sensitive, shot-noise-limited, and ruggedized Raman signal acquisition using a time-correlated photon-counting system. Compared with conventional Raman spectrometers, over 95% weight, 65% energy consumption, and 70% cost could be removed through this design. This technique allows space- and UAV-based Raman spectrometers to robustly perform hyperspectral Raman acquisitions without excessive energy consumption.

Single-Photon Tracking for High-Speed Vision.

Quanta Imager Sensors provide photon detections at high frame rates, with negligible read-out noise, making them ideal for high-speed optical tracking. At the basic level of bit-planes or binary maps of photon detections, objects may present limited detail. However, through motion estimation and spatial reassignment of photon detections, the objects can be reconstructed with minimal motion artefacts. We here present the first demonstration of high-speed two-dimensional (2D) tracking and reconstruction of rigid, planar objects with a Quanta Image Sensor, including a demonstration of depth-resolved tracking.

Characterization of a Time-Resolved Diffuse Optical Spectroscopy Prototype Using Low-Cost, Compact Single Photon Avalanche Detectors for Tissue Optics Applications.

Time-resolved diffuse optical spectroscopy (TR-DOS) is an increasingly used method to determine the optical properties of diffusive media, particularly for medical applications including functional brain, breast and muscle measurements. For medical imaging applications, important features of new generation TR-DOS systems are low-cost, small size and efficient inverse modeling. To address the issues of low-cost, compact size and high integration capabilities, we have developed free-running (FR) single-photon avalanche diodes (SPADs) using 130 nm silicon complementary metal-oxide-semiconductor (CMOS) technology and used it in a TR-DOS prototype. This prototype was validated using assessments from two known protocols for evaluating TR-DOS systems for tissue optics applications. Following the basic instrumental performance protocol, our prototype had sub-nanosecond total instrument response function and low differential non-linearity of a few percent. Also, using light with optical power lower than the maximum permissible exposure for human skin, this prototype can acquire raw data in reflectance geometry for phantoms with optical properties similar to human tissues. Following the MEDPHOT protocol, the absolute values of the optical properties for several homogeneous phantoms were retrieved with good accuracy and linearity using a best-fitting model based on the Levenberg-Marquardt method. Overall, the results of this study show that our silicon CMOS-based SPAD detectors can be used to build a multichannel TR-DOS prototype. Also, real-time functional monitoring of human tissue such as muscles, breasts and newborn heads will be possible by integrating this detector with a time-to-digital converter (TDC).