A robust and versatile platform for image scanning microscopy enabling super-resolution FLIM.

Image scanning microscopy (ISM) can improve the effective spatial resolution of confocal microscopy to its theoretical limit. However, current implementations are not robust or versatile, and are incompatible with fluorescence lifetime imaging (FLIM). We describe an implementation of ISM based on a single-photon detector array that enables super-resolution FLIM and improves multicolor, live-cell and in-depth imaging, thereby paving the way for a massive transition from confocal microscopy to ISM.

Temperature-dependent photon detection efficiency model for InGaAs/InP SPADs.

InGaAs/InP single-photon avalanche diodes (SPADs) are nowadays employed in many photon-counting applications in the near-infrared range. Photon detection efficiency (PDE) is one of the most important parameters of these detectors and here we present a model to precisely estimate it at different temperatures. Starting from optical and electrical TCAD simulations, we selected the most suitable models for the complex refractive indexes, ionization coefficients and minority carrier lifetime from the literature, and adjusted them so to include temperature and doping dependences. The good agreement between measured and simulated curves shows that our model is a valid tool to estimate PDE before device fabrication.

A Compact Multi-Distance DCS and Time Domain NIRS Hybrid System for Hemodynamic and Metabolic Measurements.

In this work, we present a new multi-distance diffuse correlation spectroscopy (DCS) device integrated with a compact state-of-the-art time domain near infrared spectroscopy (TD-NIRS) device. The hybrid DCS and TD-NIRS system allows to retrieve information on blood flow, tissue oxygenation, and oxygen metabolic rate. The DCS device performances were estimated in terms of stability, repeatability, ability in retrieving variations of diffusion coefficient, influence of the tissue optical properties, effect of varying count rates and depth sensitivity. Crosstalk between DCS and TD-NIRS optical signals was also evaluated. Finally, in vivo experiments (venous and arterial cuff occlusions on the arm) were conducted to test the ability of the hybrid system in measuring blood flow variations.

High concentration factor diffractive microlenses integrated with CMOS single-photon avalanche diode detector arrays for fill-factor improvement.

Large-format single-photon avalanche diode (SPAD) arrays often suffer from low fill-factors-the ratio of the active area to the overall pixel area. The detection efficiency of these detector arrays can be vastly increased with the integration of microlens arrays designed to concentrate incident light onto the active areas and may be refractive or diffractive in nature. The ability of diffractive optical elements (DOEs) to efficiently cover a square or rectangular pixel, combined with their capability of working as fast lenses (i.e., ∼f/3) makes them versatile and practical lens designs for use in sparse photon applications using microscale, large-format detector arrays. Binary-mask-based photolithography was employed to fabricate fast diffractive microlenses for two designs of 32×32 SPAD detector arrays, each design having a different pixel pitch and fill-factor. A spectral characterization of the lenses is performed, as well as analysis of performance under different illumination conditions from wide- to narrow-angle illumination (i.e., f/2 to f/22 optics). The performance of the microlenses presented exceeds previous designs in terms of both concentration factor (i.e., increase in light collection capability) and lens speed. Concentration factors greater than 33× are achieved for focal lengths in the substrate material as short as 190µm, representing a microlens f-number of 3.8 and providing a focal spot diameter of <4µm. These results were achieved while retaining an extremely high degree of performance uniformity across the 1024 devices in each case, which demonstrates the significant benefits to be gained by the implementation of DOEs as part of an integrated detector system using SPAD arrays with very small active areas.

0.16 µm⁻BCD Silicon Photomultipliers with Sharp Timing Response and Reduced Correlated Noise.

Silicon photomultipliers (SiPMs) have improved significantly over the last years and now are widely employed in many different applications. However, the custom fabrication technologies exploited for commercial SiPMs do not allow the integration of any additional electronics, e.g., on-chip readout and analog (or digital) processing circuitry. In this paper, we present the design and characterization of two microelectronics-compatible SiPMs fabricated in a 0.16 µm⁻BCD (Bipolar-CMOS-DMOS) technology, with 0.67 mm × 0.67 mm total area, 10 × 10 square pixels and 53% fill-factor (FF). The photon detection efficiency (PDE) surpasses 33% (FF included), with a dark-count rate (DCR) of 330 kcps. Although DCR density is worse than that of state-of-the-art SiPMs, the proposed fabrication technology enables the development of cost-effective systems-on-chip (SoC) based on SiPM detectors. Furthermore, correlated noise components, i.e., afterpulsing and optical crosstalk, and photon timing response are comparable to those of best-in-class commercial SiPMs.

Towards correcting atmospheric beam wander via pump beam control in a down conversion process.

Correlated photon pairs produced by a spontaneous parametric down conversion (SPDC) process can be used for secure quantum communication over long distances including free space transmission over a link through turbulent atmosphere. We experimentally investigate the possibility to utilize the intrinsic strong correlation between the pump and output photon spatial modes to mitigate the negative targeting effects of atmospheric beam wander. Our approach is based on a demonstration observing the deflection of the beam on a spatially resolved array of single photon avalanche diodes (SPAD-array).

Non-line-of-sight imaging using a time-gated single photon avalanche diode.

By using time-of-flight information encoded in multiply scattered light, it is possible to reconstruct images of objects hidden from the camera's direct line of sight. Here, we present a non-line-of-sight imaging system that uses a single-pixel, single-photon avalanche diode (SPAD) to collect time-of-flight information. Compared to earlier systems, this modification provides significant improvements in terms of power requirements, form factor, cost, and reconstruction time, while maintaining a comparable time resolution. The potential for further size and cost reduction of this technology make this system a good base for developing a practical system that can be used in real world applications.

Enhanced single-photon time-of-flight 3D ranging.

We developed a system for acquiring 3D depth-resolved maps by measuring the Time-of-Flight (TOF) of single photons. It is based on a CMOS 32 × 32 array of Single-Photon Avalanche Diodes (SPADs) and 350 ps resolution Time-to-Digital Converters (TDCs) into each pixel, able to provide photon-counting or photon-timing frames every 10 µs. We show how such a system can be used to scan large scenes in just hundreds of milliseconds. Moreover, we show how to exploit TDC unwarping and refolding for improving signal-to-noise ratio and extending the full-scale depth range. Additionally, we merged 2D and 3D information in a single image, for easing object recognition and tracking.

Fill-factor improvement of Si CMOS single-photon avalanche diode detector arrays by integration of diffractive microlens arrays.

Single-photon avalanche diode (SPAD) detector arrays generally suffer from having a low fill-factor, in which the photo-sensitive area of each pixel is small compared to the overall area of the pixel. This paper describes the integration of different configurations of high efficiency diffractive optical microlens arrays onto a 32 × 32 SPAD array, fabricated using a 0.35 µm CMOS technology process. The characterization of SPAD arrays with integrated microlens arrays is reported over the spectral range of 500-900 nm, and a range of f-numbers from f/2 to f/22. We report an average concentration factor of 15 measured for the entire SPAD array with integrated microlens array. The integrated SPAD and microlens array demonstrated a very high uniformity in overall efficiency.

Fast silicon photomultiplier improves signal harvesting and reduces complexity in time-domain diffuse optics.

We present a proof of concept prototype of a time-domain diffuse optics probe exploiting a fast Silicon PhotoMultiplier (SiPM), featuring a timing resolution better than 80 ps, a fast tail with just 90 ps decay time-constant and a wide active area of 1 mm2. The detector is hosted into the probe and used in direct contact with the sample under investigation, thus providing high harvesting efficiency by exploiting the whole SiPM numerical aperture and also reducing complexity by avoiding the use of cumbersome fiber bundles. Our tests also demonstrate high accuracy and linearity in retrieving the optical properties and suitable contrast and depth sensitivity for detecting localized inhomogeneities. In addition to a strong improvement in both instrumentation cost and size with respect to legacy solutions, the setup performances are comparable to those of state-of-the-art time-domain instrumentation, thus opening a new way to compact, low-cost and high-performance time-resolved devices for diffuse optical imaging and spectroscopy.

Diffuse optics using a dual window fast-gated counter.

In this paper we demonstrate the advantages of a fast-gated counter in achieving high count-rate and reducing costs of timing equipment in a time-resolved diffuse optical spectroscopy setup. We experimentally prove the equivalence between the fast-gated counter we developed and a traditional time-correlated single-photon counting setup in terms of depth sensitivity and signal-to-noise ratio. Additionally, we show the suitability of this device for bilayer analysis and to estimate the absorption coefficient of homogeneous diffusing media. Finally, we present a proof-of-principle arterial occlusion measurement on a healthy volunteer to validate the proposed approach in a real application. Fast-gated counters can dramatically reduce both costs and complexity in time-resolved multichannel systems, while achieving high count-rate, thus offering a great advantage in applications like brain and muscle functional imaging.

Erratum: Assessment of power spectral density of microvascular hemodynamics in skeletal muscles at very low and low-frequency via near-infrared diffuse optical spectroscopies: erratum.

[This corrects the article on p. 5994 in vol. 14, PMID: 38021143.].

Non-Invasive Monitoring of Microvascular Oxygenation and Reactive Hyperemia using Hybrid, Near-Infrared Diffuse Optical Spectroscopy for Critical Care.

The detection of levels of impairment in microvascular oxygen consumption and reactive hyperemia is vital in critical care. However, there are no practical means for a robust and quantitative evaluation. This paper describes a protocol to evaluate these impairments using a hybrid near-infrared diffuse optical device. The device contains modules for near-infrared time-resolved and diffuse correlation spectroscopies and pulse-oximetry. These modules allow the non-invasive, continuous, and real-time measurement of the absolute, microvascular blood/tissue oxygen saturation (StO2) and the blood flow index (BFI) along with the peripheral arterial oxygen saturation (SpO2). This device uses an integrated, computer-controlled tourniquet system to execute a standardized protocol with optical data acquisition from the brachioradialis muscle. The standardized vascular occlusion test (VOT) takes care of the variations in the occlusion duration and pressure reported in the literature, while the automation minimizes inter-operator differences. The protocol we describe focuses on a 3-min occlusion period but the details described in this paper can readily be adapted to other durations and cuff pressures, as well as other muscles. The inclusion of an extended baseline and post-occlusion recovery period measurement allows the quantification of the baseline values for all the parameters and the blood/tissue deoxygenation rate that corresponds to the metabolic rate of oxygen consumption. Once the cuff is released, we characterize the tissue reoxygenation rate, magnitude, and duration of the hyperemic response in BFI and StO2. These latter parameters correspond to the quantification of the reactive hyperemia, which provides information about the endothelial function. Furthermore, the above-mentioned measurements of the absolute concentration of oxygenated and deoxygenated hemoglobin, BFI, the derived metabolic rate of oxygen consumption, StO2, and SpO2 provide a yet-to-be-explored rich data set that can exhibit disease severity, personalized therapeutics, and management interventions.

Kilometer-range depth imaging at 1,550 nm wavelength using an InGaAs/InP single-photon avalanche diode detector.

We have used an InGaAs/InP single-photon avalanche diode detector module in conjunction with a time-of-flight depth imager operating at a wavelength of 1550 nm, to acquire centimeter resolution depth images of low signature objects at stand-off distances of up to one kilometer. The scenes of interest were scanned by the transceiver system using pulsed laser illumination with an average optical power of less than 600 µW and per-pixel acquisition times of between 0.5 ms and 20 ms. The fiber-pigtailed InGaAs/InP detector was Peltier-cooled and operated at a temperature of 230 K. This detector was used in electrically gated mode with a single-photon detection efficiency of about 26% at a dark count rate of 16 kilocounts per second. The system's overall instrumental temporal response was 144 ps full width at half maximum. Measurements made in daylight on a number of target types at ranges of 325 m, 910 m, and 4.5 km are presented, along with an analysis of the depth resolution achieved.

In vivocharacterization of the optical and hemodynamic properties of the human sternocleidomastoid muscle through ultrasound-guided hybrid near-infrared spectroscopies.

Objective.In this paper, we present a detailedin vivocharacterization of the optical and hemodynamic properties of the human sternocleidomastoid muscle (SCM), obtained through ultrasound-guided near-infrared time-domain and diffuse correlation spectroscopies.Approach.A total of sixty-five subjects (forty-nine females, sixteen males) among healthy volunteers and thyroid nodule patients have been recruited for the study. Their SCM hemodynamic (oxy-, deoxy- and total hemoglobin concentrations, blood flow, blood oxygen saturation and metabolic rate of oxygen extraction) and optical properties (wavelength dependent absorption and reduced scattering coefficients) have been measured by the use of a novel hybrid device combining in a single unit time-domain near-infrared spectroscopy, diffuse correlation spectroscopy and simultaneous ultrasound imaging.Main results.We provide detailed tables of the results related to SCM baseline (i.e. muscle at rest) properties, and reveal significant differences on the measured parameters due to variables such as side of the neck, sex, age, body mass index, depth and thickness of the muscle, allowing future clinical studies to take into account such dependencies.Significance.The non-invasive monitoring of the hemodynamics and metabolism of the sternocleidomastoid muscle during respiration became a topic of increased interest partially due to the increased use of mechanical ventilation during the COVID-19 pandemic. Near-infrared diffuse optical spectroscopies were proposed as potential practical monitors of increased recruitment of SCM during respiratory distress. They can provide clinically relevant information on the degree of the patient's respiratory effort that is needed to maintain an optimal minute ventilation, with potential clinical application ranging from evaluating chronic pulmonary diseases to more acute settings, such as acute respiratory failure, or to determine the readiness to wean from invasive mechanical ventilation.

Assessment of power spectral density of microvascular hemodynamics in skeletal muscles at very low and low-frequency via near-infrared diffuse optical spectroscopies.

In this work, we used a hybrid time domain near-infrared spectroscopy (TD-NIRS) and diffuse correlation spectroscopy (DCS) device to retrieve hemoglobin and blood flow oscillations of skeletal muscle microvasculature. We focused on very low (VLF) and low-frequency (LF) oscillations (i.e., frequency lower than 0.145 Hz), that are related to myogenic, neurogenic and endothelial activities. We measured power spectral density (PSD) of blood flow and hemoglobin concentration in four muscles (thenar eminence, plantar fascia, sternocleidomastoid and forearm) of 14 healthy volunteers to highlight possible differences in microvascular hemodynamic oscillations. We observed larger PSDs for blood flow compared to hemoglobin concentration, in particular in case of distal muscles (i.e., thenar eminence and plantar fascia). Finally, we compared the PSDs measured on the thenar eminence of healthy subjects with the ones measured on a septic patient in the intensive care unit: lower power in the endothelial-dependent frequency band, and larger power in the myogenic ones were observed in the septic patient, in accordance with previous works based on laser doppler flowmetry.

Single-fiber diffuse optical time-of-flight spectroscopy.

We demonstrate interstitial diffuse optical time-of-fight spectroscopy based on a single fiber for both light delivery and detection. Detector saturation due to the massive short-time reflection is avoided by ultrafast gating of a single photon avalanche diode. We show that the effects of scattering and absorption are separable and that absorption can be assessed independently of scattering. Measurements on calibrated liquid phantoms and subsequent Monte Carlo-based evaluation illustrate that absorption coefficients can be accurately assessed over a wide range of medically relevant optical properties. Our findings pave the way to simplified and less invasive interstitial in vivo spectroscopy.

Multi-laboratory performance assessment of diffuse optics instruments: the BitMap exercise.

SIGNIFICANCE: Multi-laboratory initiatives are essential in performance assessment and standardization-crucial for bringing biophotonics to mature clinical use-to establish protocols and develop reference tissue phantoms that all will allow universal instrument comparison. AIM: The largest multi-laboratory comparison of performance assessment in near-infrared diffuse optics is presented, involving 28 instruments and 12 institutions on a total of eight experiments based on three consolidated protocols (BIP, MEDPHOT, and NEUROPT) as implemented on three kits of tissue phantoms. A total of 20 synthetic indicators were extracted from the dataset, some of them defined here anew. APPROACH: The exercise stems from the Innovative Training Network BitMap funded by the European Commission and expanded to include other European laboratories. A large variety of diffuse optics instruments were considered, based on different approaches (time domain/frequency domain/continuous wave), at various stages of maturity and designed for different applications (e.g., oximetry, spectroscopy, and imaging). RESULTS: This study highlights a substantial difference in hardware performances (e.g., nine decades in responsivity, four decades in dark count rate, and one decade in temporal resolution). Agreement in the estimates of homogeneous optical properties was within 12% of the median value for half of the systems, with a temporal stability of <5 % over 1 h, and day-to-day reproducibility of <3 % . Other tests encompassed linearity, crosstalk, uncertainty, and detection of optical inhomogeneities. CONCLUSIONS: This extensive multi-laboratory exercise provides a detailed assessment of near-infrared Diffuse optical instruments and can be used for reference grading. The dataset-available soon in an open data repository-can be evaluated in multiple ways, for instance, to compare different analysis tools or study the impact of hardware implementations.

Fast-gated single-photon counting technique widens dynamic range and speeds up acquisition time in time-resolved measurements.

In many time-domain single-photon measurements, wide dynamic range (more than 5 orders of magnitude) is required in short acquisition time (few seconds). We report on the results of a novel technique based on a time-gated Single-Photon Avalanche Diode (SPAD) able to increase the dynamic range of optical investigations. The optical signal is acquired only in well-defined time intervals. Very fast 200-ps gate-ON transition is used to avoid the undesired strong signal, which can saturate the detector, hide the fainter useful signal and reduce the dynamic range. In experimental measurements, we obtained a dynamic range approaching 8 decades in few minutes of acquisition.

Confocal-based fluorescence fluctuation spectroscopy with a SPAD array detector.

The combination of confocal laser-scanning microscopy (CLSM) and fluorescence fluctuation spectroscopy (FFS) is a powerful tool in studying fast, sub-resolution biomolecular processes in living cells. A detector array can further enhance CLSM-based FFS techniques, as it allows the simultaneous acquisition of several samples-essentially images-of the CLSM detection volume. However, the detector arrays that have previously been proposed for this purpose require tedious data corrections and preclude the combination of FFS with single-photon techniques, such as fluorescence lifetime imaging. Here, we solve these limitations by integrating a novel single-photon-avalanche-diode (SPAD) array detector in a CLSM system. We validate this new implementation on a series of FFS analyses: spot-variation fluorescence correlation spectroscopy, pair-correlation function analysis, and image-derived mean squared displacement analysis. We predict that the unique combination of spatial and temporal information provided by our detector will make the proposed architecture the method of choice for CLSM-based FFS.