Fluorescence lifetime imaging with a single-photon SPAD array using long overlapping gates: an experimental and theoretical study.

Fluorescence lifetime imaging microscopy (FLIM) of the metabolic co-enzyme nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] is a popular method to monitor single-cell metabolism within unperturbed, living 3D systems. However, FLIM of NAD(P)H has not been performed in a light-sheet geometry, which is advantageous for rapid imaging of cells within live 3D samples. We aim to design, validate, and demonstrate a proof-of-concept light-sheet system for NAD(P)H FLIM. A single-photon avalanche diode camera was integrated into a light-sheet microscope to achieve optical sectioning and limit out-of-focus contributions for NAD(P)H FLIM of single cells. An NAD(P)H light-sheet FLIM system was built and validated with fluorescence lifetime standards and with time-course imaging of metabolic perturbations in pancreas cancer cells with 10s integration times. NAD(P)H light-sheet FLIM in vivo was demonstrated with live neutrophil imaging in a larval zebrafish tail wound also with 10s integration times. Finally, the theoretical and practical imaging speeds for NAD(P)H FLIM were compared across laser scanning and light-sheet geometries, indicating a to acquisition speed advantage for the light sheet compared to the laser scanning geometry. FLIM of NAD(P)H is feasible in a light-sheet geometry and is attractive for 3D live cell imaging applications, such as monitoring immune cell metabolism and migration within an organism.

A time-gated 4×128 Single Photon Avalanche Diode (SPAD) line array with a 512-channel Time-to-digital converter (TDC) has been designed and fabricated in a 0.35 μm high voltage CMOS technology for pulsed Raman spectroscopy. In Raman spectroscopy the SPAD array can be designed as a line array with a high fill factor because electronics can be designed on either side of the SPAD array. In this paper cross talk measurements of the array have been carried out to investigate the probability of the cross talk between adjacent SPADs in an array with high density. Measurements showed a cross talk probability of 0.11% with a pitch of adjacent SPADs of 32 μm. In addition, the largest probability of the cross talk was measured to occur with a short delay.

Protein kinases of the microtubule affinity regulating kinase (MARK)/Par-1 family play important roles in the establishment of cellular polarity, cell cycle control, and intracellular signal transduction. Disturbance of their function is linked to cancer and brain diseases, e.g. lissencephaly and Alzheimer disease. To understand the biological role of MARK family kinases, we searched for specific inhibitors and a biosensor for MARK activity. A screen of the ChemBioNet library containing ~18,000 substances yielded several compounds with inhibitory activity in the low micromolar range and capable of inhibiting MARK activity in cultured cells and primary neurons, as judged by MARK-dependent phosphorylation of microtubule-associated proteins and its consequences for microtubule integrity. Four of the compounds share a 9-oxo-9H-acridin-10-yl structure as a basis that will serve as a lead for optimization of inhibition efficiency. To test these inhibitors, we developed a cellular biosensor for MARK activity based on a MARK target sequence attached to the 14-3-3 scaffold protein and linked to enhanced cyan or teal and yellow fluorescent protein as FRET donor and acceptor pairs. Transfection of the teal/yellow fluorescent protein sensor into neurons and imaging by fluorescence lifetime imaging revealed that MARK was particularly active in the axons and growth cones of differentiating neurons.

Chapter 3 Time domain FLIM: Theory, instrumentation, and data analysis

The integrated circuit design of Single Photon Avalanche Diode (SPAD) with quenching circuit in CMOS is highly desirable photon detection at high rates, but low counting rates constrain image acquisition rates and dynamic range. The characterization should primarily define the dead time of quenching SPAD circuit in order to estimate the SPAD performance prior fabrication. This paper reports the development and characterization of the mathematical model SPAD on passively quenched SPAD circuit with ballast resistor. An improved model in defining the dead time (tD) response for SPAD is used to characterize the performance of 8×1 passively quenched SPAD array. The time response for both quenching and recharging time are developed for 180 nm depletion layer which means low voltage technology. Hence that, the 8×1 passively SPAD array circuit is designed by using Silterra 180nm CMOS technology for uncorrelated time measurements with on-chip 4-bit counter to improve the counting rate. The passive quenching circuit design on-chip would enable the capability to perform at higher speed, which is more than 100 kHz. In addition, are presented in this paper.

In recent years, time-correlated single photon counting (TCSPC) has become the technique of choice in many life science analyses, where fast and faint luminous signals are recorded with picosecond accuracy. Nevertheless, the maximum operating frequency of a single TCSPC acquisition channel limits the measurement speed, especially when scanning point systems are exploited. In order to increase the speed of TCSPC experiments, many multichannel systems based on single photon avalanche diode arrays have been proposed in the literature, which integrate thousands of pixels on the same chip. Unfortunately, the huge number of data generated by this kind of system can easily bring to the saturation of the transfer bandwidth to the external processing unit. For this reason, several different readout architectures have been proposed in the literature, attempting to exploit at best the limited bandwidth under TCSPC operating conditions. In this paper, some typical readout approaches, namely clock-driven and event-driven readouts, are discussed and compared, along with a recently-introduced router-based algorithm that is specifically designed to obtain maximum bandwidth exploitation under any condition. Quantitative comparisons are performed starting from imager response of the systems, which is the rate of recorded events in the case of uniform illumination of the detector array.

In this paper we present a novel single-photon detector specifically designed for Non-Line-Of-Sight (NLOS) imaging applications within the framework of the DARPA REVEAL program. The instrument is based on a linear 16 × 1 Complementary Metal-Oxide-Semiconductor (CMOS) Single-Photon Avalanche Diode (SPAD) array operated in fast-gated mode by a novel fast-gating Active Quenching Circuit (AQC) array, which enables the detectors with sub-ns transitions thanks to a SPAD-dummy approach. The detector exhibits a timing resolution better than 50 ps (Full Width at Half Maximum - FWHM) at a measurement repetition rate up to 40 MHz, and provides 16 independent outputs compatible with commercial Time-Correlated Single-Photon Counting (TCSPC) instrumentation. The instrument has been experimentally characterized and operated in preliminary NLOS imaging acquisitions where a 40 × 60 cm hidden object is successfully reconstructed by scanning over a grid of 150 × 150 positions.

We report a time-correlated single-photon counting (TCSPC) imaging system based on a line-scanning architecture. The system benefits from the high fill-factor, active area, and large dimension of an advanced CMOS single-photon avalanche diode (SPAD) array line-sensor. A two-dimensional image is constructed using a moving mirror to scan the line-sensor field-of-view (FOV) across the target, to enable the efficient acquisition of a two-dimensional 0.26 Mpixel TCSPC image. We demonstrate the capabilities of the system for TCSPC imaging and locating objects obscured in scattering media - specifically to locate a series of discrete point sources of light along an optical fibre submerged in a highly scattering solution. We demonstrate that by selectively imaging using early arriving photons which have undergone less scattering than later arriving photons, our TCSPC imaging system is able to locate the position of discrete point sources of light than a non-time-resolved imaging system.

We report on the design and characterization of a multipurpose 64 × 32 CMOS single-photon avalanche diode (SPAD) array. The chip is fabricated in a high-voltage 0.35-μm CMOS technology and consists of 2048 pixels, each combining a very low noise (100 cps at 5-V excess bias) 30-μm SPAD, a prompt avalanche sensing circuit, and digital processing electronics. The array not only delivers two-dimensional intensity information through photon counting in either free-running (down to 10-μs integration time) or time-gated mode, but can also perform smart light demodulation with in-pixel background suppression. The latter feature enables phase-resolved imaging for extracting either three-dimensional depth-resolved images or decay lifetime maps, by measuring the phase shift between a modulated excitation light and the reflected photons. Pixel-level memories enable fully parallel processing and global-shutter readout, preventing motion artifacts (e.g., skew, wobble, motion blur) and partial exposure effects. The array is able to acquire very fast optical events at high frame-rate (up to 100 000 fps) and at single-photon level. Low-noise SPADs ensure high dynamic range (up to 110 dB at 100 fps) with peak photon detection efficiency of almost 50% at 410 nm. The SPAD imager provides different operating modes, thus, enabling both time-domain applications, like fluorescence lifetime imaging (FLIM) and fluorescence correlation spectroscopy, as well as frequency-domain FLIM and lock-in 3-D ranging for automotive vision and lidar.

Time-resolved non-line-of-sight (NLOS) imaging based on single-photon avalanche diode (SPAD) detectors have demonstrated impressive results in recent years. To acquire adequate number of indirect photons from a hidden scene in the presence of overwhelming amount of early-arrival photons, a single-gated SPAD is widely employed to mitigate pile-up. However, additional prior knowledge of the hidden range and relay surface profile are required to preset the gating position and implement the reconstruction. With this work, we propose a gradient-gated technique to alleviate these shortcomings (pile-up and prior knowledge) in NLOS imaging with the development of a 6 × 6 SPAD array fabricated in a standard 55-nm Bipolar-CMOS-DMOS (BCD) technology. The SPAD sensor includes a delay-locked loop (DLL) block, a gating generator and a pixel array, which can be flexibly configured to free-running, single-gating and gradient-gating modes. The rise time of the gates is less than 450 ps (from 10% to 90%). In gradient-gating mode, the interval time between adjacent gates can be configured from 300 ps to 2 ns. In single-gating mode, the minimum gate window is measured below 5 ns. We demonstrated the sensor in a confocal NLOS imaging system that reconstructs hidden scenes with both retroreflective and diffuse objects. The presented scheme enables the development of calibration-free NLOS imaging modality for practical applications.

Single-Photon Avalanche Diodes (SPADs) are an emerging pixel technology able to detect the arrival of single photons. Arrays of SPADs can be used to image a scene in binary frames, indicating whether there has been a photon detection in a pixel during a very short frame exposure (e.g., few microseconds). Like many imaging technologies, SPAD sensors suffer from false photon hits, called Dark Count (DC), which increase the photon count, resulting in abnormally bright pixels, called hot pixels. Correction methods used for common CCD/CMOS sensors, such as dark frame subtraction, are ineffective for SPADs, which have non-linear response in observed counts due to their 1-bit quantization. Thus, we derive an analytical model of the count bias introduced by DC, which is signal-dependent, and provide a pixel-wise, closed-form DC correction algorithm. Remarkably, our correction is optimal in expectation, meaning that on average it removes perfectly the distortion. We validate our method on real SPAD acquisitions and synthetic data, showing that it significantly improves image reconstruction.

We report the development of a novel line-scanning microscope capable of acquiring high-speed time-correlated single-photon counting (TCSPC)-based fluorescence lifetime imaging microscopy (FLIM) imaging. The system consists of a laser-line focus, which is optically conjugated to a 1024 × 8 single-photon avalanche diode (SPAD)-based line-imaging complementary metal-oxide semiconductor (CMOS), with 23.78 µm pixel pitch at 49.31% fill factor. Incorporation of on-chip histogramming on the line-sensor enables acquisition rates 33 times faster than our previously reported bespoke high-speed FLIM platforms. We demonstrate the imaging capability of the high-speed FLIM platform in a number of biological applications.

Fluorescence lifetime imaging microscopy (FLIM) provides valuable quantitative insights into fluorophores' chemical microenvironment. Due to long computation times and the lack of accessible, open-source real-time analysis toolkits, traditional analysis of FLIM data, particularly with the widely used time-correlated single-photon counting (TCSPC) approach, typically occurs after acquisition. As a result, uncertainties about the quality of FLIM data persist even after collection, frequently necessitating the extension of imaging sessions. Unfortunately, prolonged sessions not only risk missing important biological events but also cause photobleaching and photodamage. We present the first open-source program designed for real-time FLIM analysis during specimen scanning to address these challenges. Our approach combines acquisition with real-time computational and visualization capabilities, allowing us to assess FLIM data quality on the fly. Our open-source real-time FLIM viewer, integrated as a Napari plugin, displays phasor analysis and rapid lifetime determination (RLD) results computed from real-time data transmitted by acquisition software such as the open-source Micro-Manager-based OpenScan package. Our method facilitates early identification of FLIM signatures and data quality assessment by providing preliminary analysis during acquisition. This not only speeds up the imaging process, but it is especially useful when imaging sensitive live biological samples.

Quantum imaging and microscopy profit from entangled photons to surpass the boundaries of classical optics, thus improving image resolution. Thanks to their single-photon sensitivity, readout noise absence, low-voltage operation and high frame-rate, detectors based on Single-Photon Avalanche-Diodes (SPADs) are particularly suited for this application field. We discuss strengths and weaknesses of different SPAD based architectures (classified in SiPMs, SPAD arrays, or SiPM arrays), highlighting those to be exploited as quantum imagers. As just SPAD arrays are capable of spatial resolution at single-SPAD level and, through the possible implementation of quantum-specific on-chip processing, we identified them as the forefront detector type for quantum imaging and microscopy.

This paper outlines an experimental demonstration of a Bayesian image reconstruction approach to achieve rapid single-photon color imaging of moving objects. The capacity to extract the color of objects is important in a variety of target identification and computer vision applications. Nonetheless, it remains challenging to achieve high-speed color imaging of moving objects in low-photon flux environments. The low-photon regime presents particular challenges for efficient spectral separation and identification, while unsupervised image reconstruction algorithms are often slow and computationally expensive. In this paper, we address both of these difficulties using a combination of hardware and computational solutions. We demonstrate color imaging using a Single-Photon Avalanche Diode (SPAD) detector array for rapid, low-light-level data acquisition, with an integrated color filter array (CFA) for efficient spectral unmixing. High-speed image reconstruction is achieved using a bespoke Bayesian algorithm to produce high-fidelity color videos. The analysis is conducted first on simulated data allowing different pixel formats and photon flux scenarios to be investigated. Experiments are then performed using a plasmonic metasurface-based CFA, integrated with a 64 × 64 pixel format SPAD array. Passive imaging is conducted using white-light illumination of multi-colored, moving targets. Intensity information is recorded in a series of 2D photon-counting SPAD frames, from which accurate color information is extracted using the fast Bayesian method introduced herein. The per-frame reconstruction rate proves to be hundreds of times faster than the previous computational method. Furthermore, this approach yields additional information in the form of uncertainty measures, which can be used to assist with imaging system optimization and decision-making in real-world applications. The techniques demonstrated point the way towards rapid video-rate single-photon color imaging. The developed Bayesian algorithm, along with more advanced SPAD technology and utilization of time-correlated single-photon counting (TCSPC) will permit live 3D, color videography in extremely low-photon flux environments.