Subsurface fluorescence time-of-flight imaging using a large-format single-photon avalanche diode sensor for tumor depth assessment.

Significance: Fluorescence guidance is used clinically by surgeons to visualize anatomical and/or physiological phenomena in the surgical field that are difficult or impossible to detect by the naked eye. Such phenomena include tissue perfusion or molecular phenotypic information about the disease being resected. Conventional fluorescence-guided surgery relies on long, microsecond scale laser pulses to excite fluorescent probes. However, this technique only provides two-dimensional information; crucial depth information, such as the location of malignancy below the tissue surface, is not provided. Aim: We developed a depth sensing imaging technique using light detection and ranging (LiDAR) time-of-flight (TOF) technology to sense the depth of target tissue while overcoming the influence of tissue optical properties and fluorescent probe concentration. Approach: The technology is based on a large-format (512×512 pixel), binary, gated, single-photon avalanche diode (SPAD) sensor with an 18 ps time-gate step, synchronized with a picosecond pulsed laser. The fast response of the sensor was developed and tested for its ability to quantify fluorescent inclusions at depth and optical properties in tissue-like phantoms through analytical model fitting of the fast temporal remission data. Results: After calibration and algorithmic extraction of the data, the SPAD LiDAR technique allowed for sub-mm resolution depth sensing of fluorescent inclusions embedded in tissue-like phantoms, up to a maximum of 5 mm in depth. The approach provides robust depth sensing even in the presence of variable tissue optical properties and separates the effects of fluorescence depth from absorption and scattering variations. Conclusions: LiDAR TOF fluorescence imaging using an SPAD camera provides both fluorescence intensity images and the temporal profile of fluorescence, which can be used to determine the depth at which the signal is emitted over a wide field of view. The proposed tool enables fluorescence imaging at a higher depth in tissue and with higher spatial precision than standard, steady-state fluorescence imaging tools, such as intensity-based near-infrared fluorescence imaging, optical coherence tomography, Raman spectroscopy, or confocal microscopy. Integration of this technique into a standard surgical tool could enable rapid, more accurate estimation of resection boundaries, thereby improving the surgeon's efficacy and efficiency, and ultimately improving patient outcomes.

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

SwissSPAD3 is the latest of a family of widefield time-gated SPAD imagers developed for fluorescence lifetime imaging (FLI) applications. Its distinctive features are (i) the ability to define shorter gates than its predecessors (width W < 1 ns), (ii) support for laser repetition rates up to at least 80 MHz and (iii) a dual-gate architecture providing an effective duty cycle of 100%. We present widefield macroscopic FLI measurements of short lifetime NIR dyes, analyzed using the phasor approach. The results are compared with those previously obtained with SwissSPAD2 and to theoretical predictions.

Correlated-photon imaging at 10 volumetric images per second.

The correlation properties of light provide an outstanding tool to overcome the limitations of traditional imaging techniques. A relevant case is represented by correlation plenoptic imaging (CPI), a quantum-inspired volumetric imaging protocol employing spatio-temporally correlated photons from either entangled or chaotic sources to address the main limitations of conventional light-field imaging, namely, the poor spatial resolution and the reduced change of perspective for 3D imaging. However, the application potential of high-resolution imaging modalities relying on photon correlations is limited, in practice, by the need to collect a large number of frames. This creates a gap, unacceptable for many relevant tasks, between the time performance of correlated-light imaging and that of traditional imaging methods. In this article, we address this issue by exploiting the photon number correlations intrinsic in chaotic light, combined with a cutting-edge ultrafast sensor made of a large array of single-photon avalanche diodes (SPADs). This combination of source and sensor is embedded within a novel single-lens CPI scheme enabling to acquire 10 volumetric images per second. Our results place correlated-photon imaging at a competitive edge and prove its potential in practical applications.

Challenges and prospects for multi-chip microlens imprints on front-side illuminated SPAD imagers.

The overall sensitivity of frontside-illuminated, silicon single-photon avalanche diode (SPAD) arrays has often suffered from fill factor limitations. The fill factor loss can however be recovered by employing microlenses, whereby the challenges specific to SPAD arrays are represented by large pixel pitch (> 10 µm), low native fill factor (as low as ∼10%), and large size (up to 10 mm). In this work we report on the implementation of refractive microlenses by means of photoresist masters, used to fabricate molds for imprints of UV curable hybrid polymers deposited on SPAD arrays. Replications were successfully carried out for the first time, to the best of our knowledge, at wafer reticle level on different designs in the same technology and on single large SPAD arrays with very thin residual layers (∼10 µm), as needed for better efficiency at higher numerical aperture (NA > 0.25). In general, concentration factors within 15-20% of the simulation results were obtained for the smaller arrays (32×32 and 512×1), achieving for example an effective fill factor of 75.6-83.2% for a 28.5 µm pixel pitch with a native fill factor of 28%. A concentration factor up to 4.2 was measured on large 512×512 arrays with a pixel pitch of 16.38 µm and a native fill factor of 10.5%, whereas improved simulation tools could give a better estimate of the actual concentration factor. Spectral measurements were also carried out, resulting in good and uniform transmission in the visible and NIR.

Massively parallel, real-time multispeckle diffuse correlation spectroscopy using a 500 × 500 SPAD camera.

Diffuse correlation spectroscopy (DCS) is a promising noninvasive technique for monitoring cerebral blood flow and measuring cortex functional activation tasks. Taking multiple parallel measurements has been shown to increase sensitivity, but is not easily scalable with discrete optical detectors. Here we show that with a large 500 × 500 SPAD array and an advanced FPGA design, we achieve an SNR gain of almost 500 over single-pixel mDCS performance. The system can also be reconfigured to sacrifice SNR to decrease correlation bin width, with 400 ns resolution being demonstrated over 8000 pixels.

LinoSPAD2: an FPGA-based, hardware-reconfigurable 512×1 single-photon camera system.

We report on LinoSPAD2, a single-photon camera system, comprising a 512×1 single-photon avalanche diode (SPAD) front-end and one or two FPGA-based back-ends. Digital signals generated by the SPADs are processed by the FPGA in real time, whereas the FPGA offers full reconfigurability at a very high level of granularity both in time and space domains. The LinoSPAD2 camera system can process 512 SPADs simultaneously through 256 channels, duplicated on each FPGA-based back-end, with a bank of 64 time-to-digital converters (TDCs) operating at 133 MSa/s, whereas each TDC has a time resolution of 20 ps (LSB). To the best of our knowledge, LinoSPAD2 is the first fully reconfigurable SPAD camera system of large format. The SPAD front-end features a pitch of 26.2 µm, a native fill factor of 25.1%, and a microlens array achieving 2.3× concentration factor. At room temperature, the median dark count rate (DCR) is 80 cps at 7 V excess bias, the peak photon detection probability (PDP) is 53% at 520 nm wavelength, and the single-photon timing resolution (SPTR) is 50 ps FWHM. The instrument response function (IRF) is around 100 ps FWHM at system level. The LinoSPAD2 camera system is suitable for numerous applications, including LiDAR imaging, heralded spectroscopy, compressive Raman sensing, and other computational imaging techniques.

In vitro and in vivo NIR fluorescence lifetime imaging with a time-gated SPAD camera.

Near-infrared (NIR) fluorescence lifetime imaging (FLI) provides a unique contrast mechanism to monitor biological parameters and molecular events in vivo. Single-photon avalanche diode (SPAD) cameras have been recently demonstrated in FLI microscopy (FLIM) applications, but their suitability for in vivo macroscopic FLI (MFLI) in deep tissues remains to be demonstrated. Herein, we report in vivo NIR MFLI measurement with SwissSPAD2, a large time-gated SPAD camera. We first benchmark its performance in well-controlled in vitro experiments, ranging from monitoring environmental effects on fluorescence lifetime, to quantifying Förster resonant energy transfer (FRET) between dyes. Next, we use it for in vivo studies of target-drug engagement in live and intact tumor xenografts using FRET. Information obtained with SwissSPAD2 was successfully compared to that obtained with a gated intensified charge-coupled device (ICCD) camera, using two different approaches. Our results demonstrate that SPAD cameras offer a powerful technology for in vivo preclinical applications in the NIR window.

Light detection and ranging with entangled photons.

Single-photon light detection and ranging (LiDAR) is a key technology for depth imaging through complex environments. Despite recent advances, an open challenge is the ability to isolate the LiDAR signal from other spurious sources including background light and jamming signals. Here we show that a time-resolved coincidence scheme can address these challenges by exploiting spatio-temporal correlations between entangled photon pairs. We demonstrate that a photon-pair-based LiDAR can distill desired depth information in the presence of both synchronous and asynchronous spurious signals without prior knowledge of the scene and the target object. This result enables the development of robust and secure quantum LiDAR systems and paves the way to time-resolved quantum imaging applications.

Resolving the Controversy in Biexciton Binding Energy of Cesium Lead Halide Perovskite Nanocrystals through Heralded Single-Particle Spectroscopy.

Understanding exciton-exciton interaction in multiply excited nanocrystals is crucial to their utilization as functional materials. Yet, for lead halide perovskite nanocrystals, which are promising candidates for nanocrystal-based technologies, numerous contradicting values have been reported for the strength and sign of their exciton-exciton interaction. In this work, we unambiguously determine the biexciton binding energy in single cesium lead halide perovskite nanocrystals at room temperature. This is enabled by the recently introduced single-photon avalanche diode array spectrometer, capable of temporally isolating biexciton-exciton emission cascades while retaining spectral resolution. We demonstrate that CsPbBr3 nanocrystals feature an attractive exciton-exciton interaction, with a mean biexciton binding energy of 10 meV. For CsPbI3 nanocrystals, we observe a mean biexciton binding energy that is close to zero, and individual nanocrystals show either weakly attractive or weakly repulsive exciton-exciton interaction. We further show that, within ensembles of both materials, single-nanocrystal biexciton binding energies are correlated with the degree of charge-carrier confinement.

Theoretical minimum uncertainty of single-molecule localizations using a single-photon avalanche diode array.

Single-photon avalanche diode (SPAD) arrays can be used for single-molecule localization microscopy (SMLM) because of their high frame rate and lack of readout noise. SPAD arrays have a binary frame output, which means photon arrivals should be described as a binomial process rather than a Poissonian process. Consequentially, the theoretical minimum uncertainty of the localizations is not accurately predicted by the Poissonian Cramér-Rao lower bound (CRLB). Here, we derive a binomial CRLB and benchmark it using simulated and experimental data. We show that if the expected photon count is larger than one for all pixels within one standard deviation of a Gaussian point spread function, the binomial CRLB gives a 46% higher theoretical uncertainty than the Poissonian CRLB. For typical SMLM photon fluxes, where no saturation occurs, the binomial CRLB predicts the same uncertainty as the Poissonian CRLB. Therefore, the binomial CRLB can be used to predict and benchmark localization uncertainty for SMLM with SPAD arrays for all practical emitter intensities.

Heralded Spectroscopy Reveals Exciton-Exciton Correlations in Single Colloidal Quantum Dots.

Multiply excited states in semiconductor quantum dots feature intriguing physics and play a crucial role in nanocrystal-based technologies. While photoluminescence provides a natural probe to investigate these states, room-temperature single-particle spectroscopy of their emission has proved elusive due to the temporal and spectral overlap with emission from the singly excited and charged states. Here, we introduce biexciton heralded spectroscopy enabled by a single-photon avalanche diode array based spectrometer. This allows us to directly observe biexciton-exciton emission cascades and measure the biexciton binding energy of single quantum dots at room temperature, even though it is well below the scale of thermal broadening and spectral diffusion. Furthermore, we uncover correlations hitherto masked in ensembles of the biexciton binding energy with both charge-carrier confinement and fluctuations of the local electrostatic potential. Heralded spectroscopy has the potential of greatly extending our understanding of charge-carrier dynamics in multielectron systems and of parallelization of quantum optics protocols.

Wide-field time-gated SPAD imager for phasor-based FLIM applications.

We describe the performance of a new wide area time-gated single-photon avalanche diode (SPAD) array for phasor-FLIM, exploring the effect of gate length, gate number and signal intensity on the measured lifetime accuracy and precision. We conclude that the detector functions essentially as an ideal shot noise limited sensor and is capable of video rate FLIM measurement. The phasor approach used in this work appears ideally suited to handle the large amount of data generated by this type of very large sensor (512 × 512 pixels), even in the case of small number of gates and limited photon budget.

Single-Photon, Time-Gated, Phasor-Based Fluorescence Lifetime Imaging through Highly Scattering Medium.

Fluorescence lifetime imaging (FLI) is increasingly recognized as a powerful tool for biochemical and cellular investigations, including in vivo applications. Fluorescence lifetime is an intrinsic characteristic of any fluorescent dye which, to a large extent, does not depend on excitation intensity and signal level. In particular, it allows distinguishing dyes with similar emission spectra, offering additional multiplexing capabilities. However, in vivo FLI in the visible range is complicated by the contamination by (i) tissue autofluorescence, which decreases contrast, and by (ii) light scattering and absorption in tissues, which significantly reduce fluorescence intensity and modify the temporal profile of the signal. Here, we demonstrate how these issues can be accounted for and overcome, using a new time-gated single-photon avalanche diode array camera, SwissSPAD2, combined with phasor analysis to provide a simple and fast visual method for lifetime imaging. In particular, we show how phasor dispersion increases with increasing scattering and/or decreasing fluorescence intensity. Next, we show that as long as the fluorescence signal of interest is larger than the phantom autofluorescence, the presence of a distinct lifetime can be clearly identified with appropriate background correction. We use these results to demonstrate the detection of A459 cells expressing the fluorescent protein mCyRFP1 through highly scattering and autofluorescent phantom layers. These results showcase the possibility to perform FLI in challenging conditions, using standard, bright, visible fluorophore or fluorescence proteins.

A 512×512 SPAD Image Sensor with Integrated Gating for Widefield FLIM.

We report on SwissSPAD2, an image sensor with 512×512 photon-counting pixels, each comprising a single-photon avalanche diode (SPAD), a 1-bit memory, and a gating mechanism capable of turning the SPAD on and off, with a skew of 250ps and 344ps, respectively, for a minimum duration of 5.75ns. The sensor is designed to achieve a frame rate of up to 97,700 binary frames per second and sub-40ps gate shifts. By synchronizing it with a pulsed laser and using multiple successive overlapping gates, one can reconstruct a molecule's fluorescent response with picosecond temporal resolution. Thanks to the sensor's number of pixels (the largest to date) and the fully integrated gated operation, SwissSPAD2 enables widefield FLIM with an all-solid-state solution and at relatively high frame rates. This was demonstrated with preliminary results on organic dyes and semiconductor quantum dots using both decay fitting and phasor analysis. Furthermore, pixels with an exceptionally low dark count rate and high photon detection probability enable uniform and high quality imaging of biologically relevant fluorescent samples stained with multiple dyes. While future versions will feature the addition of microlenses and optimize firmware speed, our results open the way to low-cost alternatives to commercially available scientific time-resolved imagers.

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

Developing large arrays of single-photon avalanche diodes (SPADs) with on-chip time-correlated single-photon counting (TCSPC) capabilities continues to be a difficult task due to stringent silicon real estate constraints, high data rates and system complexity. As an alternative to TCSPC, time-gated architectures have been proposed, where the numbers of photons detected within different time gates are used as a replacement to the usual time-resolved luminescence decay. However, because of technological limitations, the minimum gate length implement is on the order of nanoseconds, longer than most fluorophore lifetimes of interest. However, recent FLIM measurements have shown that it is mainly the gate step and rise/fall time, rather than its length, which determine lifetime resolution. In addition, the large number of photons captured by longer gates results in higher SNR. In this paper, we study the effects of using long, overlapping gates on lifetime extraction by phasor analysis, using a recently developed 512×512 time-gated SPAD array. The experiments used Cy3B, Rhodamine 6G and Atto550 dyes as test samples. The gate window length was varied between 11.3 ns and 23 ns while the gate step was varied between 17.86 ps and 3 ns. We validated the results with a standard TCSPC setup and investigated the case of multi-exponential samples through simulations. Results indicate that lifetime extraction is not degraded by the use of longer gates, nor is the ability to resolve multi-exponential decays.

Phasor-based widefield FLIM using a gated 512×512 single-photon SPAD imager.

Single-photon avalanche diode (SPAD) imagers can perform fast time-resolved imaging in a compact form factor, by exploiting the processing capability and speed of integrated CMOS electronics. Developments in SPAD imagers have recently made them compatible with widefield microscopy, thanks to array formats approaching one megapixel and sensitivity and noise levels approaching those of established technologies. In this paper, phasor-based FLIM is demonstrated with a gated binary 512×512 SPAD imager, which can operate with a gate length as short as 5.75 ns, a minimum gate step of 17.9 ps, and up to 98 kfps readout rate (1-bit frames). Lifetimes of ATTO 550 and Rhodamine 6G (R6G) solutions were measured across a 472×256 sub-array using phasor analysis, acquiring data by shifting a 13.1 ns gate window across the 50 ns laser period. The measurement accuracy obtained when employing such a scheme based on long, overlapping gates was validated by comparison with TCSPC measurements and fitting analysis results based on a standard Levenberg-Marquardt algorithm (>90% accuracy for the lifetime of R6G and ATTO 550). This demonstrates the ability of the proposed method to measure short lifetimes without minimum gate length requirements. The FLIM frame rate of the overall system can be increased up to a few fps for phasor-based widefield FLIM (moving closer to real-time operation) by FPGA-based parallel computation with continuous acquisition at the full speed of 98 kfps.