NIR Fluorescence lifetime macroscopic imaging with a time-gated SPAD camera.

The performance of SwissSPAD2 (SS2), a large scale, widefield time-gated CMOS SPAD imager developed for fluorescence lifetime imaging, has recently been described in the context of visible range and fluorescence lifetime imaging microscopy (FLIM) of dyes with lifetimes in the 2.5 - 4 ns range. Here, we explore its capabilities in the NIR regime relevant for small animal imaging, where its sensitivity is lower and typical NIR fluorescent dye lifetimes are much shorter (1 ns or less). We carry out this study in a simple macroscopic imaging setup based on a compact NIR picosecond pulsed laser, an engineered diffuser-based illumination optics, and NIR optimized imaging lens suitable for well-plate or small animal imaging. Because laser repetition rates can vary between models, but the synchronization signal frequency accepted by SS2 is fixed to 20 MHz, we first checked that a simple frequency-division scheme enables data recording for different laser repetition rates. Next, we acquired data using different time gate widths, including gates with duration longer than the laser period, and analyzed the resulting data using both standard nonlinear least-square fit (NLSF) and phasor analysis. We show that the fixed synchronization rate and large gate widths characterizing SS2 (10 ns and over) are not an obstacle to accurately extracting lifetime in the 1 ns range and to distinguishing between close lifetimes. In summary, SS2 and similar very large gated SPAD imagers appear as a versatile alternative to other widefield time-resolved detectors for NIR fluorescence lifetime imaging, including preclinical molecular applications.

In Phantom Validation of Time-Domain Near-Infrared Optical Tomography Pioneer for Imaging Brain Hypoxia and Hemorrhage.

The neonatal brain is a vulnerable organ, and lesions due to hemorrhage and/or ischemia occur frequently in preterm neonates. Even though neuroprotective therapies exist, there is no tool available to detect the ischemic lesions. To address this problem, we have recently designed and built the new time-domain near-infrared optical tomography (TD NIROT) system - Pioneer. Here we present the results of a phantom study of the system performance. We used silicone phantoms to mimic risky situations for brain lesions: hemorrhage and hypoxia. Employing Pioneer, we were able to reconstruct accurately both position and optical properties of these inhomogeneities.

Probe Design Optimization for Time-Domain NIROT "Pioneer" System for Imaging the Oxygenation of the Preterm Brain.

In preterm infants, there is a risk of life-lasting impairments due to hemorrhagic/ischemic lesions. Our time-domain (TD) near-infrared optical tomography (NIROT) system "Pioneer" aims at detecting both disorders with high spatial resolution. Successfully tested on phantoms, "Pioneer" entered the phase of improvements and enhancements. The current probe (A-probe) was adapted for an optoacoustics instrument. A new probe (B-probe) optimized for TD measurements is required. Our aim is to determine the optimal arrangement of light sources in the B-probe to increase the sensitivity and the resolution of Pioneer and to improve the ability of the system to detect both ischemia and hemorrhage. To do this, we simulated TD-NIROT signals in NIRFAST, a MATLAB-based package used to model near-infrared light propagation through tissue. We used 16 × 16 detector array, with ~2.2 mm distance between the detectors. Light sources were arranged around the field of view (FoV). We performed forward simulations of light propagation through a "homogeneous case" (HC) tissue (µ's = 5.6 cm-1, µa = 0.07 cm-1). Next, we simulated light propagation through "inhomogeneous case" -tissue' (IC) tissue by adding ischemia (µa = µa · 2.5 cm-1) or hemorrhage (µa = µa · 50 cm-1) to HT as a spherical inclusion of 5 mm radius at different depths in the FoV center and identified the source location that provides the higher contrast on the FoV: maxi ∈ I (FoVContrastSOURCE). It was found that sources located closer to the FoV center generate greater contrast for late photons. This study suggests the light sources in B-probe should be closer to the FoV center. The higher sensitivity is expected to lead to a higher image quality.

Time-Resolved NIROT 'Pioneer' System for Imaging Oxygenation of the Preterm Brain: Preliminary Results.

In preterm infants, there is a risk of long-term cognitive, motor and behavioral impairments due to hemorrhagic and/or ischemic lesions. If detected early, lesions can be prevented. A bedside imaging modality, capable of early detection of both disorders, is necessary. We present the state of development of a tomographic imager (named Pioneer), that will be capable of determining the oxygenation of the preterm-infant brain with high spatial resolution. Pioneer is a time-resolved near-infrared optical tomography (TR NIROT) instrument. It employs multiple wavelength laser light in short pulses on 11 distinct locations and measures the re-emerging light in a contactless fashion by means of a time-correlated single-photon counting (TCSPC) camera (named Piccolo) covering ~4.9 cm2 with 300 detectors. Timing response of the entire system is 116 ps. An in-house designed biocompatible source ring ensures fixed relative positions of sources and detectors and provides a secure interface between the patient and the probe. At the present state, the NIROT Pioneer system successfully detected a 6x6x50 mm3 inclusion 3 cm deep inside a phantom. These results confirm that the Pioneer imager is working as expected and is on a solid path towards full 3D tissue oxygenation imaging.

Compact solid-state CMOS single-photon detector array for in vivo NIR fluorescence lifetime oncology measurements.

In near infrared fluorescence-guided surgical oncology, it is challenging to distinguish healthy from cancerous tissue. One promising research avenue consists in the analysis of the exogenous fluorophores' lifetime, which are however in the (sub-)nanosecond range. We have integrated a single-photon pixel array, based on standard CMOS SPADs (single-photon avalanche diodes), in a compact, time-gated measurement system, named FluoCam. In vivo measurements were carried out with indocyanine green (ICG)-modified derivatives targeting the αvß 3 integrin, initially on a genetically engineered mouse model of melanoma injected with ICG conjugated with tetrameric cyclic pentapeptide (ICG-E[c(RGD f K)4]), then on mice carrying tumour xenografts of U87-MG (a human primary glioblastoma cell line) injected with monomeric ICG-c(RGD f K). Measurements on tumor, muscle and tail locations allowed us to demonstrate the feasibility of in vivo lifetime measurements with the FluoCam, to determine the characteristic lifetimes (around 500 ps) and subtle lifetime differences between bound and unbound ICG-modified fluorophores (10% level), as well as to estimate the available photon fluxes under realistic conditions.

Single-photon imaging in complementary metal oxide semiconductor processes.

This paper describes the basics of single-photon counting in complementary metal oxide semiconductors, through single-photon avalanche diodes (SPADs), and the making of miniaturized pixels with photon-counting capability based on SPADs. Some applications, which may take advantage of SPAD image sensors, are outlined, such as fluorescence-based microscopy, three-dimensional time-of-flight imaging and biomedical imaging, to name just a few. The paper focuses on architectures that are best suited to those applications and the trade-offs they generate. In this context, architectures are described that efficiently collect the output of single pixels when designed in large arrays. Off-chip readout circuit requirements are described for a variety of applications in physics, medicine and the life sciences. Owing to the dynamic nature of SPADs, designs featuring a large number of SPADs require careful analysis of the target application for an optimal use of silicon real estate and of limited readout bandwidth. The paper also describes the main trade-offs involved in architecting such chips and the solutions adopted with focus on scalability and miniaturization.

An implementation of a spike-response model with escape noise using an avalanche diode.

This paper introduces a novel probabilistic spike-response model through the combination of avalanche diode-generated Poisson distributed noise, and a standard exponential decay-based spike-response curve. The noise source, which is derived from a 0.35-µm single-photon avalanche diode (kept in the dark), was tested experimentally to verify its characteristics, before being combined with a field-programmable gate-array implementation of a spike-response model. This simple model was then analyzed, and shown to reproduce seven of eight behaviors recorded during an extensive study of the ventral medial hypothalamic (VMH) region of the brain. It is thought that many of the cell types found within the VMH are fed from a tonic noise synaptic input, where the patterns generated are a product of their spike response and not their interconnection. This paper shows how this tonic noise source can be modelled, and due to the independent nature of the noise sources, provides an avenue for the exploration of networks of noise-fueled neurons, which play a significant role in pattern generation within the brain.

On the application of a monolithic array for detecting intensity-correlated photons emitted by different source types.

It is not widely appreciated that many subtleties are involved in the accurate measurement of intensity-correlated photons; even for the original experiments of Hanbury Brown and Twiss (HBT). Using a monolithic 4 x 4 array of single-photon avalanche diodes (SPADs), together with an off-chip algorithm for processing streaming data, we investigate the difficulties of measuring second-order photon correlations g((2))(x(iota), t(iota),x, t) in a wide variety of light fields that exhibit dramatically different correlation statistics: a multimode He-Ne laser, an incoherent intensity-modulated lamp-light source and a thermal light source. Our off-chip algorithm treats multiple photon-arrivals at pixel-array pairs, in any observation interval, with photon fluxes limited by detector saturation, in such a way that a correctly normalized g((2)) function is guaranteed. The impact of detector background correlations between SPAD pixels and afterpulsing effects on second-order coherence measurements is discussed. These results demonstrate that our monolithic SPAD array enables access to effects that are otherwise impossible to measure with stand-alone detectors.

Fast-fluorescence dynamics in nonratiometric calcium indicators.

The fluorescence decay of high-affinity nonratiometric Ca2+ indicator Oregon Green BAPTA-1 (OGB-1) is analyzed with unprecedented temporal resolution in the two-photon excitation regime. A triple exponential decay is shown to best fit the fluorescence dynamics of OGB-1. We provide a model for accurate measurements of the free Ca2+ concentration and dissociation constants of nonratiometric calcium indicators.