Quantifying distortion in time-correlated single photon counting: a universal parameter.

One major drawback of the classic time-correlated single photon counting (TCSPC) technique is pileup-related distortion. To keep it under a reasonable level, the maximum count rate has to be reduced, posing a serious limitation to the overall measurement speed. This means that there is an intrinsic trade-off between speed and distortion: either count rate is raised, but distortion is worsened, or distortion is minimized at the expense of speed. In both cases, it is impossible to precisely evaluate the degree of distortion introduced. Here comes our new, to the best of our knowledge, figure of merit, which is able to provide a numerical estimate of the distortion whatever the signal shape is, marking a real turning point in the way of doing TCSPC. In this article, this new parameter will be defined and its effectiveness will be demonstrated by means of mathematical computations.

Timing measurements with silicon single photon avalanche diodes: principles and perspectives [Invited].

Picosecond timing of single photons has laid the foundation of a great variety of applications, from life sciences to quantum communication, thanks to the combination of ultimate sensitivity with a bandwidth that cannot be reached by analog recording techniques. Nowadays, more and more applications could still be enabled or advanced by progress in the available instrumentation, resulting in a steadily increasing research interest in this field. In this scenario, single-photon avalanche diodes (SPADs) have gained a key position, thanks to the remarkable precision they are able to provide, along with other key advantages like ruggedness, compactness, large signal amplitude, and room temperature operation, which neatly distinguish them from other solutions like superconducting nanowire single-photon detectors and silicon photomultipliers. With this work, we aim at filling a gap in the literature by providing a thorough discussion of the main design rules and tradeoffs for silicon SPADs and the electronics employed along them to achieve high timing precision. In the end, we conclude with our outlook on the future by summarizing new routes that could benefit from present and prospective timing features of silicon SPADs.

4 ns dead time with a fully integrated active quenching circuit driving a custom single photon avalanche diode.

At the present time, Single Photon Avalanche Diodes (SPADs) are the enabling devices in many applications, ranging from medical imaging to laser ranging and from remote sensing to quantum key distribution. Even though they belong to different scientific domains, these applications share the need for a detector capable of attaining high count rates possibly without trading it off with other key detector's features, such as afterpulsing probability, photon detection efficiency, and dark counts. In this work, we present the characterization of a fast integrated active quenching circuit capable of driving high-performance external custom-technology SPADs for single photon detection in the visible wavelength range. Combining the prompt intervention of the electronic circuitry and the performance of a custom-technology SPAD, we attained count rates up to 250 MCps while keeping the afterpulsing probability within 2%.

Above pile-up fluorescence microscopy with a 32 Mc/s single-channel time-resolved SPAD system.

One of the major drawbacks of time-correlated single-photon counting (TCSPC) is generally represented by pile-up distortion, which strongly bounds the maximum acquisition speed to a few percent of the laser excitation rate. Based on a previous theoretical analysis, recently we presented the first, to the best of our knowledge, low-distortion and high-speed TCSPC system capable of overcoming the pile-up limitation by perfectly matching the single-photon avalanche diode (SPAD) dead time to the laser period. In this work, we validate the proposed system in a standard fluorescence measurement by comparing experimental data with the reference theoretical framework. As a result, a count rate of 32 Mc/s was achieved with a single-channel system still observing a negligible lifetime distortion.

Custom-Technology Single-Photon Avalanche Diode Linear Detector Array for Underwater Depth Imaging.

We present an optical depth imaging system suitable for highly scattering underwater environments. The system used the time-correlated single-photon counting (TCSPC) technique and the time-of-flight approach to obtain depth profiles. The single-photon detection was provided by a linear array of single-photon avalanche diode (SPAD) detectors fabricated in a customized silicon fabrication technology for optimized efficiency, dark count rate, and jitter performance. The bi-static transceiver comprised a pulsed laser diode source with central wavelength 670 nm, a linear array of 16 × 1 Si-SPAD detectors, with a dedicated TCSPC acquisition module. Cylindrical lenses were used to collect the light scattered by the target and image it onto the sensor. These laboratory-based experiments demonstrated single-photon depth imaging at a range of 1.65 m in highly scattering conditions, equivalent up to 8.3 attenuation lengths between the system and the target, using average optical powers of up to 15 mW. The depth and spatial resolution of this sensor were investigated in different scattering conditions.

Custom silicon technology for SPAD-arrays with red-enhanced sensitivity and low timing jitter.

Single-photon detection is an invaluable tool for many applications ranging from basic research to consumer electronics. In this respect, the Single Photon Avalanche Diode (SPAD) plays a key role in enabling a broad diffusion of these techniques thanks to its remarkable performance, room-temperature operation, and scalability. In this paper we present a silicon technology that allows the fabrication of SPAD-arrays with an unprecedented combination of low timing jitter (95 ps FWHM) and high detection efficiency at red and near infrared wavelengths (peak of 70% at 650 nm, 45% at 800 nm). We discuss the device structure, the fabrication process, and we present a thorough experimental characterization of the fabricated detectors. We think that these results can pave the way to new exciting developments in many fields, ranging from quantum optics to single molecule spectroscopy.

Multispectral compressive fluorescence lifetime imaging microscopy with a SPAD array detector.

Multispectral/hyperspectral fluorescence lifetime imaging microscopy (λFLIM) is a promising tool for studying functional and structural biological processes. The rich information content provided by a multidimensional dataset is often in contrast with the acquisition speed. In this work, we develop and experimentally demonstrate a wide-field λFLIM setup, based on a novel time-resolved 18×1 single-photon avalanche diode array detector working in a single-pixel camera scheme, which parallelizes the spectral detection, reducing measurement time. The proposed system, which implements a single-pixel camera with a compressive sensing scheme, represents an optimal microscopy framework towards the design of λFLIM setups.

Fast fully-integrated front-end circuit to overcome pile-up limits in time-correlated single photon counting with single photon avalanche diodes.

Time-Correlated Single Photon Counting (TCSPC) is an essential tool in many scientific applications, where the recording of optical pulses with picosecond precision is required. Unfortunately, a key issue has to be faced: distortion phenomena can affect TCSPC experiments at high count rates. In order to avoid this problem, TCSPC experiments have been commonly carried out by limiting the maximum operating frequency of a measurement channel below 5% of the excitation frequency, leading to a long acquisition time. Recently, it has been demonstrated that matching the detector dead time to the excitation period allows to keep distortion around zero regardless of the rate of impinging photons. This solution paves the way to unprecedented measurement speed in TCSPC experiments. In this scenario, the front-end circuits that drive the detector play a crucial role in determining the performance of the system, both in terms of measurement speed and timing performance. Here we present two fully integrated front-end circuits for Single Photon Avalanche Diodes (SPADs): a fast Active Quenching Circuit (AQC) and a fully-differential current pick-up circuit. The AQC can apply very fast voltage variations, as short as 1.6ns, to reset external custom-technology SPAD detectors. A fast reset, indeed, is a key parameter to maximize the measurement speed. The current pick-up circuit is based on a fully differential structure which allows unprecedented rejection of disturbances that typically affect SPAD-based systems at the end of the dead time. The circuit permits to sense the current edge resulting from a photon detection with picosecond accuracy and precision even a few picoseconds after the end of the dead time imposed by the AQC. This is a crucial requirement when the system is operated at high rates. Both circuits have been deeply characterized, especially in terms of achievable measurement speed and timing performance.

48-spot single-molecule FRET setup with periodic acceptor excitation.

Single-molecule Förster resonance energy transfer (smFRET) allows measuring distances between donor and acceptor fluorophores on the 3-10 nm range. Solution-based smFRET allows measurement of binding-unbinding events or conformational changes of dye-labeled biomolecules without ensemble averaging and free from surface perturbations. When employing dual (or multi) laser excitation, smFRET allows resolving the number of fluorescent labels on each molecule, greatly enhancing the ability to study heterogeneous samples. A major drawback to solution-based smFRET is the low throughput, which renders repetitive measurements expensive and hinders the ability to study kinetic phenomena in real-time. Here we demonstrate a high-throughput smFRET system that multiplexes acquisition by using 48 excitation spots and two 48-pixel single-photon avalanche diode array detectors. The system employs two excitation lasers allowing separation of species with one or two active fluorophores. The performance of the system is demonstrated on a set of doubly labeled double-stranded DNA oligonucleotides with different distances between donor and acceptor dyes along the DNA duplex. We show that the acquisition time for accurate subpopulation identification is reduced from several minutes to seconds, opening the way to high-throughput screening applications and real-time kinetics studies of enzymatic reactions such as DNA transcription by bacterial RNA polymerase.

Red-Enhanced Photon Detection Module Featuring a 32 × 1 Single-Photon Avalanche Diode Array.

In this letter, the development and the experimental characterization of a new photon detection module, based on a 32×1 red-enhanced single-photon avalanche diode (RE-SPAD) array, are presented. A custom-developed technology has been exploited to design a detector having large-area pixels (50-µm diameter) with optimized performance. With an excess bias voltage Voυ = 15 V, a photon detection efficiency as high as 57% at 600 nm (33% at 800 nm) is achieved, along with dark count rate in the kHz range and optical crosstalk probability as low as 0.29%. The remarkable detection efficiency of the RE-SPAD array makes the module particularly suitable for all applications where high detection efficiency in the red/near-infrared range is mandatory. As an example, the performance of the array module is demonstrated to match the demanding requirements of multispot single-molecule fluorescence spectroscopy.

Optical crosstalk in SPAD arrays for high-throughput single-molecule fluorescence spectroscopy.

Single-molecule fluorescence spectroscopy (SMFS), based on the detection of individual molecules freely diffusing through the excitation spot of a confocal microscope, has allowed unprecedented insights into biological processes at the molecular level, but suffers from limited throughput. We have recently introduced a multispot version of SMFS, which allows achieving high-throughput SMFS by virtue of parallelization, and relies on custom silicon single-photon avalanche diode (SPAD) detector arrays. Here, we examine the premise of this parallelization approach, which is that data acquired from different spots is uncorrelated. In particular, we measure the optical crosstalk characteristics of the two 48-pixel SPAD arrays used in our recent SMFS studies, and demonstrate that it is negligible (crosstalk probability ≤ 1.1 10-3) and undetectable in cross-correlation analysis of actual single-molecule fluorescence data.

Development and characterization of an 8×8 SPAD-array module for gigacount per second applications.

In recent years the development of Single-Photon Avalanche Diodes (SPADs) had a big impact on single-photon counting applications requiring high-performance detectors in terms of Dark Count Rate (DCR), Photon Detection Efficiency (PDE), afterpulsing probability, etc. Among these, it is possible to find applications in single-molecule fluorescence spectroscopy that suffer from long-time measurements. In these cases SPAD arrays can be a solution in order to shorten the measurement time, thanks to the high grade of parallelism they can provide. Moreover, applications in other fields (e.g. astronomy) demand for large-area single-photon detectors, able also to handle very high count rates. For these reasons we developed a new single-photon detection module, featuring an 8 × 8 SPAD array. Thanks to a dedicated silicon technology, the performance of the detector have been finely optimized, reaching a 49% detection efficiency at 550 nm, as well as low dark counts (2 kcount/s maximum all over the array). This module can be used in two different modes: the first is a multi-spot configuration, allowing the acquisition of 64 optical signals at the same time and considerably reducing the time needed for a measurement. The second operation mode instead exploits all the pixels in a combined mode, allowing the detection of a 64-times higher maximum photon rate (up to 2 Gcount/s). In addition, this configuration provides also an extended dynamic range and allows to attain photon number resolving capabilities. Dark counts, detection efficiency, linearity, afterpulsing and crosstalk probability have been characterized at different operating conditions.

16-Ch Time-resolved Single-Molecule Spectroscopy Using Line Excitation.

Single-molecule spectroscopy on freely-diffusing molecules allows detecting conformational changes of biomolecules without perturbation from surface immobilization. Resolving fluorescence lifetimes increases the sensitivity in detecting conformational changes and overcomes artifacts common in intensity-based measurements. Common to all freely-diffusing techniques, however, are the long acquisition times. We report a time-resolved multispot system employing a 16-channel SPAD array and TCSPC electronics, which overcomes the throughput issue. Excitation is obtained by shaping a 532 nm pulsed laser into a line, matching the linear SPAD array geometry. We show that the line-excitation is a robust and cost-effective approach to implement multispot systems based on linear detector arrays.

Multispot single-molecule FRET: High-throughput analysis of freely diffusing molecules.

We describe an 8-spot confocal setup for high-throughput smFRET assays and illustrate its performance with two characteristic experiments. First, measurements on a series of freely diffusing doubly-labeled dsDNA samples allow us to demonstrate that data acquired in multiple spots in parallel can be properly corrected and result in measured sample characteristics consistent with those obtained with a standard single-spot setup. We then take advantage of the higher throughput provided by parallel acquisition to address an outstanding question about the kinetics of the initial steps of bacterial RNA transcription. Our real-time kinetic analysis of promoter escape by bacterial RNA polymerase confirms results obtained by a more indirect route, shedding additional light on the initial steps of transcription. Finally, we discuss the advantages of our multispot setup, while pointing potential limitations of the current single laser excitation design, as well as analysis challenges and their solutions.

Time-domain diffuse correlation spectroscopy.

Physiological monitoring of oxygen delivery to the brain has great significance for improving the management of patients at risk for brain injury. Diffuse correlation spectroscopy (DCS) is a rapidly growing optical technology able to non-invasively assess the blood flow index (BFi) at the bedside. The current limitations of DCS are the contamination introduced by extracerebral tissue and the need to know the tissue's optical properties to correctly quantify the BFi. To overcome these limitations, we have developed a new technology for time-resolved diffuse correlation spectroscopy. By operating DCS in the time domain (TD-DCS), we are able to simultaneously acquire the temporal point-spread function to quantify tissue optical properties and the autocorrelation function to quantify the BFi. More importantly, by applying time-gated strategies to the DCS autocorrelation functions, we are able to differentiate between short and long photon paths through the tissue and determine the BFi for different depths. Here, we present the novel device and we report the first experiments in tissue-like phantoms and in rodents. The TD-DCS method opens many possibilities for improved non-invasive monitoring of oxygen delivery in humans.

Silicon technologies for arrays of Single Photon Avalanche Diodes.

In order to fulfill the requirements of many applications, we recently developed a new technology aimed at combining the advantages of traditional thin and thick silicon Single Photon Avalanche Diodes (SPAD). In particular we demonstrated single-pixel detectors with a remarkable improvement in the Photon Detection Efficiency in the red/near-infrared spectrum (e.g. 40% at 800nm) while maintaining a timing jitter better than 100ps. In this paper we discuss the limitations of such Red-Enhanced (RE) technology from the point of view of the fabrication of small arrays of SPAD and we propose modifications to the structure aimed at overcoming these issues. We also report the first preliminary experimental results attained on devices fabricated adopting the improved structure.

High-voltage integrated active quenching circuit for single photon count rate up to 80 Mcounts/s.

Single photon avalanche diodes (SPADs) have been subject to a fast improvement in recent years. In particular, custom technologies specifically developed to fabricate SPAD devices give the designer the freedom to pursue the best detector performance required by applications. A significant breakthrough in this field is represented by the recent introduction of a red enhanced SPAD (RE-SPAD) technology, capable of attaining a good photon detection efficiency in the near infrared range (e.g. 40% at a wavelength of 800 nm) while maintaining a remarkable timing resolution of about 100ps full width at half maximum. Being planar, the RE-SPAD custom technology opened the way to the development of SPAD arrays particularly suited for demanding applications in the field of life sciences. However, to achieve such excellent performance custom SPAD detectors must be operated with an external active quenching circuit (AQC) designed on purpose. Next steps toward the development of compact and practical multichannel systems will require a new generation of monolithically integrated AQC arrays. In this paper we present a new, fully integrated AQC fabricated in a high-voltage 0.18 µm CMOS technology able to provide quenching pulses up to 50 Volts with fast leading and trailing edges. Although specifically designed for optimal operation of RE-SPAD devices, the new AQC is quite versatile: it can be used with any SPAD detector, regardless its fabrication technology, reaching remarkable count rates up to 80 Mcounts/s and generating a photon detection pulse with a timing jitter as low as 119 ps full width at half maximum. The compact design of our circuit has been specifically laid out to make this IC a suitable building block for monolithically integrated AQC arrays.

Gigacount/second Photon Detection Module Based on an 8 × 8 Single-Photon Avalanche Diode Array.

In this letter we present a compact photon detection module, based on an 8×8 array of single-photon avalanche diodes (SPADs). The use of a dedicated silicon technology for the fabrication of the sensors allows us to combine large active areas (50-µm diameter), high photon detection efficiency (49% at 550-nm wavelength) and low dark count rate. Thanks to a fully parallel architecture, the module provides voltage pulses synchronous to each photon detection for a maximum global count rate exceeding 1 Gcps. These properties makes the system suitable for operation in two different free-running modes. The first, suitable to acquire faint signals, allows multi-spot acquisitions and can be used to considerably reduce the measurement time in applications like single-molecule analysis. With the second it is possible to use all the pixels in a combined mode, to extend and move the dynamic range of the module to very high count rates and to attain number resolving capabilities.

Silicon photon-counting avalanche diodes for single-molecule fluorescence spectroscopy.

Solution-based single-molecule fluorescence spectroscopy is a powerful experimental tool with applications in cell biology, biochemistry and biophysics. The basic feature of this technique is to excite and collect light from a very small volume and work in a low concentration regime resulting in rare burst-like events corresponding to the transit of a single molecule. Detecting photon bursts is a challenging task: the small number of emitted photons in each burst calls for high detector sensitivity. Bursts are very brief, requiring detectors with fast response time and capable of sustaining high count rates. Finally, many bursts need to be accumulated to achieve proper statistical accuracy, resulting in long measurement time unless parallelization strategies are implemented to speed up data acquisition. In this paper we will show that silicon single-photon avalanche diodes (SPADs) best meet the needs of single-molecule detection. We will review the key SPAD parameters and highlight the issues to be addressed in their design, fabrication and operation. After surveying the state-of-the-art SPAD technologies, we will describe our recent progress towards increasing the throughput of single-molecule fluorescence spectroscopy in solution using parallel arrays of SPADs. The potential of this approach is illustrated with single-molecule Förster resonance energy transfer measurements.

Single-molecule FRET experiments with a red-enhanced custom technology SPAD.

Single-molecule fluorescence spectroscopy of freely diffusing molecules in solution is a powerful tool used to investigate the properties of individual molecules. Single-Photon Avalanche Diodes (SPADs) are the detectors of choice for these applications. Recently a new type of SPAD detector was introduced, dubbed red-enhanced SPAD (RE-SPAD), with good sensitivity throughout the visible spectrum and with excellent timing performance. We report a characterization of this new detector for single-molecule fluorescence resonant energy transfer (smFRET) studies on freely diffusing molecules in a confocal geometry and alternating laser excitation (ALEX) scheme. We use a series of doubly-labeled DNA molecules with donor-to-acceptor distances covering the whole range of useful FRET values. Both intensity-based (µs-ALEX) and lifetime-based (ns-ALEX) measurements are presented and compared to identical measurements performed with standard thick SPADs. Our results demonstrate the great potential of this new detector for smFRET measurements and beyond.