10-nanosecond dead time and low afterpulsing with a free-running reach-through single-photon avalanche diode.

The reduction of detector dead time represents an enabling factor in several photon counting applications. In this work, we investigate the free-running operation of reach-through single-photon avalanche diodes (SPADs) at ultra-low dead times. By employing a fast active quenching circuit with direct bonding to the detector, we are able to achieve a 10 ns dead time with a thick SPAD by Excelitas, still maintaining extremely low afterpulsing probabilities (below 1.5%).

Toward ultra-fast time-correlated single-photon counting: A compact module to surpass the pile-up limit.

Time-Correlated Single-Photon Counting (TCSPC) is an excellent technique used in a great variety of scientific experiments to acquire exceptionally fast and faint light signals. Above all, in Fluorescence Lifetime Imaging (FLIM), it is widely recognized as the gold standard to record sub-nanosecond transient phenomena with picosecond precision. Unfortunately, TCSPC has an intrinsic limitation: to avoid the so-called pile-up distortion, the experiments have been historically carried out, limiting the acquisition rate below 5% of the excitation frequency. In 2017, we demonstrated that such a limitation can be overcome if the detector dead time is exactly matched with the excitation period, thus paving the way to unprecedented speedup of FLIM measurements. In this paper, we present the first single-channel system that implements the novel proposed methodology to be used in modern TCSPC experimental setups. To achieve this goal, we designed a compact detection head, including a custom single-photon avalanche diode externally driven by a fully integrated Active Quenching Circuit (AQC), featuring a finely tunable dead time and a short reset time. The output timing signal is extracted by using a picosecond precision Pick-Up Circuit (PUC) and fed to a newly developed timing module consisting of a mixed-architecture Fast Time to Amplitude Converter (F-TAC) followed by high-performance Analog-to-Digital Converters (ADCs). Data are transmitted in real-time to a Personal Computer (PC) at USB 3.0 rate for specific and custom elaboration. Preliminary experimental results show that the new TCSPC system is suitable for implementing the proposed technique, achieving, indeed, high timing precision along with a count rate as high as 40 Mcps.

Accurate non-invasive measurement of the turn-on transition of fast gated single photon avalanche diodes.

Recently developed Active Quenching Circuits (AQCs) with fast-gating capabilities allow us to control a single photon avalanche diode with gate windows in the nanosecond and sub-nanosecond range, thus paving the way to advanced applications, especially in the field of time-correlated single photon counting. In this scenario, an accurate measurement of the time needed by the AQC to turn-on the detector is of utmost importance. Indeed, it permits us to evaluate the impact of the system in specific applications and provides a tool to designers to understand AQC limitations and to enhance its performance. Here we propose a simple non-invasive technique to accurately measure the time needed by a gated system to turn on the detector. The effectiveness of the measure has been proved on a gated system, and results have been compared to those obtained starting from the distribution of recorded photons under constant illumination, which is a widely used approach in the literature. The great advantage of the proposed approach is that it avoids typical artifacts that affect other kinds of measurements.

Note: Wide-operating-range control for thermoelectric coolers.

A new algorithm for controlling the temperature of a thermoelectric cooler is proposed. Unlike a classic proportional-integral-derivative (PID) control, which computes the bias voltage from the temperature error, the proposed algorithm exploits the linear relation that exists between the cold side's temperature and the amount of heat that is removed per unit time. Since this control is based on an existing linear relation, it is insensitive to changes in the operating point that are instead crucial in classic PID control of a non-linear system.

32-channel time-correlated-single-photon-counting system for high-throughput lifetime imaging.

Time-Correlated Single Photon Counting (TCSPC) is a very efficient technique for measuring weak and fast optical signals, but it is mainly limited by the relatively "long" measurement time. Multichannel systems have been developed in recent years aiming to overcome this limitation by managing several detectors or TCSPC devices in parallel. Nevertheless, if we look at state-of-the-art systems, there is still a strong trade-off between the parallelism level and performance: the higher the number of channels, the poorer the performance. In 2013, we presented a complete and compact 32 × 1 TCSPC system, composed of an array of 32 single-photon avalanche diodes connected to 32 time-to-amplitude converters, which showed that it was possible to overcome the existing trade-off. In this paper, we present an evolution of the previous work that is conceived for high-throughput fluorescence lifetime imaging microscopy. This application can be addressed by the new system thanks to a centralized logic, fast data management and an interface to a microscope. The new conceived hardware structure is presented, as well as the firmware developed to manage the operation of the module. Finally, preliminary results, obtained from the practical application of the technology, are shown to validate the developed system.

Note: Fully integrated active quenching circuit achieving 100 MHz count rate with custom technology single photon avalanche diodes.

The minimization of Single Photon Avalanche Diodes (SPADs) dead time is a key factor to speed up photon counting and timing measurements. We present a fully integrated Active Quenching Circuit (AQC) able to provide a count rate as high as 100 MHz with custom technology SPAD detectors. The AQC can also operate the new red enhanced SPAD and provide the timing information with a timing jitter Full Width at Half Maximum (FWHM) as low as 160 ps.

High-speed and low-distortion solution for time-correlated single photon counting measurements: A theoretical analysis.

In this paper, we describe a novel solution to increase the speed of Time-Correlated Single Photon Counting (TCSPC) measurements by almost an order of magnitude while providing, in principle, zero distortion regardless of the experimental conditions. Typically, the relatively long dead time associated with the conversion electronics requires a proper tune of the excitation power in order to avoid distortions of the reconstructed waveform due to pileup and counting loss. As a result, the maximum operating rate of a TCSPC channel is now limited between 1% and 5% of the excitation frequency, thus leading to relatively long acquisition times. We show that negligible distortion (below 1%) is guaranteed if the dead time associated with the converter is kept below the dead time of the detector, and at the same time the detector dead time is matched to the duration of the excitation period. In this way, unprecedented high-speed operation is possible. In this paper, we provide a theoretical analysis of the technique, including the main non-idealities which are introduced by a generic physical implementation. The results are supported by both numerical simulations and analytical calculations.

High-efficiency integrated readout circuit for single photon avalanche diode arrays in fluorescence lifetime imaging.

In recent years, lifetime measurements by means of the Time Correlated Single Photon Counting (TCSPC) technique have led to a significant breakthrough in medical and biological fields. Unfortunately, the many advantages of TCSPC-based approaches come along with the major drawback of a relatively long acquisition time. The exploitation of multiple channels in parallel could in principle mitigate this issue, and at the same time it opens the way to a multi-parameter analysis of the optical signals, e.g., as a function of wavelength or spatial coordinates. The TCSPC multichannel solutions proposed so far, though, suffer from a tradeoff between number of channels and performance, and the overall measurement speed has not been increased according to the number of channels, thus reducing the advantages of having a multichannel system. In this paper, we present a novel readout architecture for bi-dimensional, high-density Single Photon Avalanche Diode (SPAD) arrays, specifically designed to maximize the throughput of the whole system and able to guarantee an efficient use of resources. The core of the system is a routing logic that can provide a dynamic connection between a large number of SPAD detectors and a much lower number of high-performance acquisition channels. A key feature of our smart router is its ability to guarantee high efficiency under any operating condition.

Improving the counting efficiency in time-correlated single photon counting experiments by dead-time optimization.

Time-Correlated Single Photon Counting (TCSPC) has been long recognized as the most sensitive method for fluorescence lifetime measurements, but often requiring "long" data acquisition times. This drawback is related to the limited counting capability of the TCSPC technique, due to pile-up and counting loss effects. In recent years, multi-module TCSPC systems have been introduced to overcome this issue. Splitting the light into several detectors connected to independent TCSPC modules proportionally increases the counting capability. Of course, multi-module operation also increases the system cost and can cause space and power supply problems. In this paper, we propose an alternative approach based on a new detector and processing electronics designed to reduce the overall system dead time, thus enabling efficient photon collection at high excitation rate. We present a fast active quenching circuit for single-photon avalanche diodes which features a minimum dead time of 12.4 ns. We also introduce a new Time-to-Amplitude Converter (TAC) able to attain extra-short dead time thanks to the combination of a scalable array of monolithically integrated TACs and a sequential router. The fast TAC (F-TAC) makes it possible to operate the system towards the upper limit of detector count rate capability (∼80 Mcps) with reduced pile-up losses, addressing one of the historic criticisms of TCSPC. Preliminary measurements on the F-TAC are presented and discussed.

A 32-channel photon counting module with embedded auto/cross-correlators for real-time parallel fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) is a well-established technique to study binding interactions or the diffusion of fluorescently labeled biomolecules in vitro and in vivo. Fast FCS experiments require parallel data acquisition and analysis which can be achieved by exploiting a multi-channel Single Photon Avalanche Diode (SPAD) array and a corresponding multi-input correlator. This paper reports a 32-channel FPGA based correlator able to perform 32 auto/cross-correlations simultaneously over a lag-time ranging from 10 ns up to 150 ms. The correlator is included in a 32 × 1 SPAD array module, providing a compact and flexible instrument for high throughput FCS experiments. However, some inherent features of SPAD arrays, namely afterpulsing and optical crosstalk effects, may introduce distortions in the measurement of auto- and cross-correlation functions. We investigated these limitations to assess their impact on the module and evaluate possible workarounds.

8-Channel acquisition system for Time-Correlated Single-Photon Counting.

Nowadays, an increasing number of applications require high-performance analytical instruments capable to detect the temporal trend of weak and fast light signals with picosecond time resolution. The Time-Correlated Single-Photon Counting (TCSPC) technique is currently one of the preferable solutions when such critical optical signals have to be analyzed and it is fully exploited in biomedical and chemical research fields, as well as in security and space applications. Recent progress in the field of single-photon detector arrays is pushing research towards the development of high performance multichannel TCSPC systems, opening the way to modern time-resolved multi-dimensional optical analysis. In this paper we describe a new 8-channel high-performance TCSPC acquisition system, designed to be compact and versatile, to be used in modern TCSPC measurement setups. We designed a novel integrated circuit including a multichannel Time-to-Amplitude Converter with variable full-scale range, a D∕A converter, and a parallel adder stage. The latter is used to adapt each converter output to the input dynamic range of a commercial 8-channel Analog-to-Digital Converter, while the integrated DAC implements the dithering technique with as small as possible area occupation. The use of this monolithic circuit made the design of a scalable system of very small dimensions (95 × 40 mm) and low power consumption (6 W) possible. Data acquired from the TCSPC measurement are digitally processed and stored inside an FPGA (Field-Programmable Gate Array), while a USB transceiver allows real-time transmission of up to eight TCSPC histograms to a remote PC. Eventually, the experimental results demonstrate that the acquisition system performs TCSPC measurements with high conversion rate (up to 5 MHz/channel), extremely low differential nonlinearity (<0.04 peak-to-peak of the time bin width), high time resolution (down to 20 ps Full-Width Half-Maximum), and very low crosstalk between channels.

Development of new photon-counting detectors for single-molecule fluorescence microscopy.

Two optical configurations are commonly used in single-molecule fluorescence microscopy: point-like excitation and detection to study freely diffusing molecules, and wide field illumination and detection to study surface immobilized or slowly diffusing molecules. Both approaches have common features, but also differ in significant aspects. In particular, they use different detectors, which share some requirements but also have major technical differences. Currently, two types of detectors best fulfil the needs of each approach: single-photon-counting avalanche diodes (SPADs) for point-like detection, and electron-multiplying charge-coupled devices (EMCCDs) for wide field detection. However, there is room for improvements in both cases. The first configuration suffers from low throughput owing to the analysis of data from a single location. The second, on the other hand, is limited to relatively low frame rates and loses the benefit of single-photon-counting approaches. During the past few years, new developments in point-like and wide field detectors have started addressing some of these issues. Here, we describe our recent progresses towards increasing the throughput of single-molecule fluorescence spectroscopy in solution using parallel arrays of SPADs. We also discuss our development of large area photon-counting cameras achieving subnanosecond resolution for fluorescence lifetime imaging applications at the single-molecule level.

Avalanche current read-out circuit for low jitter parallel photon timing.

We propose a novel circuit for single photon avalanche diode (SPAD) current read-out, for photon timing applications. The circuit consists of a single transistor trans-impedance amplifier with a GHz bandwidth: the feedback loop fixes the SPAD anode voltage and allows us to obtain a high time resolution with a very high equivalent current threshold (almost 700 µA). The trans-impedance stage is followed by a low pass filter that reduces the crosstalk of other on-chip detectors and makes the designed structure suitable for multi-detector systems. The discrete components prototype presented in this letter achieves a state-of-art resolution of 34.4 ps FWHM, presents negligible crosstalk between the different pixels and opens the way for the development of an integrated structure with a large number of channels.

Custom single-photon avalanche diode with integrated front-end for parallel photon timing applications.

Emerged as a solid state alternative to photo multiplier tubes (PMTs), single-photon avalanche diodes (SPADs) are nowadays widely used in the field of single-photon timing applications. Custom technology SPADs assure remarkable performance, in particular a 10 counts/s dark count rate (DCR) at low temperature, a high photon detection efficiency (PDE) with a 50% peak at 550 nm and a 30 ps (full width at half maximum, FWHM) temporal resolution, even with large area devices, have been obtained. Over the past few years, the birth of novel techniques of analysis has led to the parallelization of the measurement systems and to a consequent increasing demand for the development of monolithic arrays of detectors. Unfortunately, the implementation of a multidimensional system is a challenging task from the electrical point of view; in particular, the avalanche current pick-up circuit, used to obtain the previously reported performance, has to be modified in order to enable high parallel temporal resolution, while minimizing the electrical crosstalk probability between channels. In the past, the problem has been solved by integrating the front-end electronics next to the photodetector, in order to reduce the parasitic capacitances and consequently the filtering action on the current signal of the SPAD, leading to an improvement of the timing jitter at higher threshold. This solution has been implemented by using standard complementary metal-oxide-semiconductor (CMOS) technologies, which, however, do not allow a complete control on the SPAD structure; for this reason the intrinsic performance of CMOS SPADs, such as DCR, PDE, and afterpulsing probability, are worse than those attainable with custom detectors. In this paper, we propose a pixel architecture, which enables the development of custom SPAD arrays in which every channel maintains the performance of the best single photodetector. The system relies on the integration of the timing signal pick-up circuit next to the photodiode, achieved by modifying the technological process flow used for the fabrication of the custom SPAD. The pixel is completed by an external standard CMOS active quenching circuit, which assures stable timing performance at quite high count rate (>1 MHz).

Parallel multispot smFRET analysis using an 8-pixel SPAD array.

Single-molecule Förster resonance energy transfer (smFRET) is a powerful tool for extracting distance information between two fluorophores (a donor and acceptor dye) on a nanometer scale. This method is commonly used to monitor binding interactions or intra- and intermolecular conformations in biomolecules freely diffusing through a focal volume or immobilized on a surface. The diffusing geometry has the advantage to not interfere with the molecules and to give access to fast time scales. However, separating photon bursts from individual molecules requires low sample concentrations. This results in long acquisition time (several minutes to an hour) to obtain sufficient statistics. It also prevents studying dynamic phenomena happening on time scales larger than the burst duration and smaller than the acquisition time. Parallelization of acquisition overcomes this limit by increasing the acquisition rate using the same low concentrations required for individual molecule burst identification. In this work we present a new two-color smFRET approach using multispot excitation and detection. The donor excitation pattern is composed of 4 spots arranged in a linear pattern. The fluorescent emission of donor and acceptor dyes is then collected and refocused on two separate areas of a custom 8-pixel SPAD array. We report smFRET measurements performed on various DNA samples synthesized with various distances between the donor and acceptor fluorophores. We demonstrate that our approach provides identical FRET efficiency values to a conventional single-spot acquisition approach, but with a reduced acquisition time. Our work thus opens the way to high-throughput smFRET analysis on freely diffusing molecules.

Cancer-testis antigen expression in triple-negative breast cancer.

BACKGROUND: cancer-testis (CT) antigens, frequently expressed in human germline cells but not in somatic tissues, may become aberrantly reexpressed in different cancer types. The aim of this study was to investigate the expression of CT antigens in breast cancer. PATIENTS AND METHODS: a total of 100 selected invasive breast cancers, including 50 estrogen receptor (ER) positive/HER2 negative and 50 triple negative (TN), were examined for NY-ESO-1 and MAGE-A expression by immunohistochemistry. RESULTS: a significantly higher expression of MAGE-A and NY-ESO-1 was detected in TN breast cancers compared with ER-positive tumors (P = 0.04). MAGE-A expression was detected in 13 (26%) TN cancers compared with 5 (10%) ER-positive tumors (P = 0.07). NY-ESO-1 expression was confirmed in nine (18%) TN tumor samples compared with two (4%) ER-positive tumors. CONCLUSIONS: MAGE-A and NY-ESO-1 CT antigens are expressed in a substantial proportion of TN breast cancers. Because of the limited therapeutic options for this group of patients, CT antigen-based vaccines might prove to be useful for patients with this phenotype of breast cancer.

New photon-counting detectors for single-molecule fluorescence spectroscopy and imaging.

Solution-based single-molecule fluorescence spectroscopy is a powerful new experimental approach with applications in all fields of natural sciences. Two typical geometries can be used for these experiments: point-like and widefield excitation and detection. In point-like geometries, the basic concept is to excite and collect light from a very small volume (typically femtoliter) and work in a concentration regime resulting in rare burst-like events corresponding to the transit of a single-molecule. Those events are accumulated over time to achieve proper statistical accuracy. Therefore the advantage of extreme sensitivity is somewhat counterbalanced by a very long acquisition time. One way to speed up data acquisition is parallelization. Here we will discuss a general approach to address this issue, using a multispot excitation and detection geometry that can accommodate different types of novel highly-parallel detector arrays. We will illustrate the potential of this approach with fluorescence correlation spectroscopy (FCS) and single-molecule fluorescence measurements. In widefield geometries, the same issues of background reduction and single-molecule concentration apply, but the duration of the experiment is fixed by the time scale of the process studied and the survival time of the fluorescent probe. Temporal resolution on the other hand, is limited by signal-to-noise and/or detector resolution, which calls for new detector concepts. We will briefly present our recent results in this domain.

Note: Fully integrated time-to-amplitude converter in Si-Ge technology.

Over the past years an always growing interest has arisen about the measurement technique of time-correlated single photon counting TCSPC), since it allows the analysis of extremely fast and weak light waveforms with a picoseconds resolution. Consequently, many applications exploiting TCSPC have been developed in several fields such as medicine and chemistry. Moreover, the development of multianode PMT and of single photon avalanche diode arrays led to the realization of acquisition systems with several parallel channels to employ the TCSPC technique in even more applications. Since TCSPC basically consists of the measurement of the arrival time of a photon, the most important part of an acquisition chain is the time measurement block, which must have high resolution and low differential nonlinearity, and in order to realize multidimensional systems, it has to be integrated to reduce both cost and area. In this paper we present a fully integrated time-to-amplitude converter, built in 0.35 µm Si-Ge technology, characterized by a good time resolution (60 ps), low differential nonlinearity (better than 3% peak to peak), high counting rate (16 MHz), low and constant power dissipation (40 mW), and low area occupation (1.38×1.28 mm(2)).

High-throughput single-molecule fluorescence spectroscopy using parallel detection.

Solution-based single-molecule fluorescence spectroscopy is a powerful new experimental approach with applications in all fields of natural sciences. The basic concept of this technique is to excite and collect light from a very small volume (typically femtoliter) and work in a concentration regime resulting in rare burst-like events corresponding to the transit of a single-molecule. Those events are accumulated over time to achieve proper statistical accuracy. Therefore the advantage of extreme sensitivity is somewhat counterbalanced by a very long acquisition time. One way to speed up data acquisition is parallelization. Here we will discuss a general approach to address this issue, using a multispot excitation and detection geometry that can accommodate different types of novel highly-parallel detector arrays. We will illustrate the potential of this approach with fluorescence correlation spectroscopy (FCS) and single-molecule fluorescence measurements obtained with different novel multipixel single-photon counting detectors.

Monolithic time to amplitude converter for time correlated single photon counting.

Time correlated single photon counting techniques allow in depth examinations of very important chemical and biological processes involving complex interactions of biomolecules. Understanding of these processes is of the utmost importance to address vital medical issues such as the origin and growth of tumors. Modern developments of fluorescence analysis techniques require compact, low cost, and high performance instrumentation, and a major path to these goals is the successful integration of the electronics. In this paper we present a fully monolithic time to amplitude converter, built in standard 0.35 microm CMOS technology, characterized by good time resolution (60 ps), low differential nonlinearity (better than 0.5% rms), short dead time (80 ns), low power dissipation (60 mW), and low area occupation (1.8x1.4 mm(2)).