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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.

Over the past few years there has been a growing interest in monolithic arrays of single photon avalanche diodes (SPAD) for spatially resolved detection of faint ultrafast optical signals. SPADs implemented in planar technologies offer the typical advantages of microelectronic devices (small size, ruggedness, low voltage, low power, etc.). Furthermore, they have inherently higher photon detection efficiency than PMTs and are able to provide, beside sensitivities down to single-photons, very high acquisition speeds. In order to make SPAD array more and more competitive in time-resolved application it is necessary to face problems like electrical crosstalk between adjacent pixel, moreover all the singlephoton timing electronics with picosecond resolution has to be developed. In this paper we present a new instrument suitable for single-photon imaging applications and made up of 32 timeresolved parallel channels. The 32x1 pixel array that includes SPAD detectors represents the system core, and an embedded data elaboration unit performs on-board data processing for single-photon counting applications. Photontiming information is exported through a custom parallel cable that can be connected to an external multichannel TCSPC system.

Over the past few years there has been a growing interest in monolithic arrays of single photon avalanche diodes (SPAD) for spatially resolved detection of faint ultrafast optical signals. SPADs implemented in planar technologies offer the typical advantages of microelectronic devices (small size, ruggedness, low voltage, low power, etc.). Furthermore, they have inherently higher photon detection efficiency than PMTs and are able to provide, beside sensitivities down to single-photons, very high acquisition speeds. Although currently available silicon devices reached remarkable performance, nevertheless further improvements are needed in order to meet the requirements of most demanding timeresolved techniques, it is necessary to face problems like electrical crosstalk between adjacent pixel, high detection efficiency in the red spectral range, large area, low dark counting rate. Moreover to develop array with high number of pixel became more and more important to develop all the TCSPC electronics with picosecond resolution to create a new family of detection system for TCSPC applications. Recent advances in our research on single photon time resolved array is here presented.

Multi-dimensional Time Correlated Single Photon Counting has nowadays reached a prominent position among analytical techniques employed in the medical and biological fields. The development of instruments able to perform simultaneously temporal and spectral fluorescence analysis (sFLIM) is limited by the performances of single-photon detectors; in fact currently available arrays cannot satisfy simultaneously all the requirements. To face this rising quest, a fully-parallel eight channel module, based on a monolithic Single Photon Avalanche Diode (SPAD) array with great temporal resolution, high Photon Detection Efficiency (PDE) and low Dark Counting Rate (DCR), has been designed and fabricated. The system relies on a novel architecture of the single pixel, based on the integration of the timing pick-up circuit next to the photodetector, making the negative effects of electrical and optical crosstalk on photon timing performance negligible. To this aim, the custom technological process used to fabricate the SPAD has been modified, allowing the integration of MOS transistors without impairing the structure and the performance of the detector. The single channel is complemented by an external Active Quenching Circuit, fabricated in a standard CMOS technology, that ensures high maximum counting rate (> 5MHz) and low afterpulsing (< 2%). Finally, the output timing signals are read and conditioned by a proper CMOS electronics. The complete pixel shows a very good temporal resolution of about 45 ps (FWHM).

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Avalanche photodiodes, which operate above the breakdown voltage in Geiger mode connected with avalanche-quenching circuits, can be used to detect single photons and are therefore called single-photon avalanche diodes SPAD’s. Circuit configurations suitable for this operation mode are critically analyzed and their relative merits in photon counting and timing applications are assessed. Simple passive-quenching circuits (PQC’s), which are useful for SPAD device testing and selection, have fairly limited application. Suitably designed active-quenching circuits (AQC’s) make it possible to exploit the best performance of SPAD’s. Thick silicon SPAD’s that operate at high voltages (250–450 V) have photon detection efficiency higher than 50% from 540- to 850-nm wavelength and still ~3% at 1064 nm. Thin silicon SPAD’s that operate at low voltages (10–50 V) have 45% efficiency at 500 nm, declining to 10% at 830 nm and to as little as 0.1% at 1064 nm. The time resolution achieved in photon timing is 20 ps FWHM with thin SPAD’s; it ranges from 350 to 150 ps FWHM with thick SPAD’s. The achieved minimum counting dead time and maximum counting rate are 40 ns and 10 Mcps with thick silicon SPAD’s, 10 ns and 40 Mcps with thin SPAD’s. Germanium and III–V compound semiconductor SPAD’s extend the range of photon-counting techniques in the near-infrared region to at least 1600-nm wavelength.

In this paper we show that single-photon avalanche detectors (SPADs) may have a strong potential as very sensitive on-wafer tools for lifetime monitoring, given their simple fabrication process and their straightforward use in the measurements. We have fabricated Si SPADs with n+–p structures and we show that dark counting has a simple exponential dependence on time, thus making it possible to extract a single lifetime parameter.

Time-correlated photon counting with solid state detectors is of considerable interest for high resolution optical time-domain reflectometry (OTDR). Up to now long measurement times were required. A new approach is described, yielding measurement times 50 times shorter and dynamic range enhanced by ≈ 20dB

A unique combination of the time-correlated photon-counting technique and single-photon avalanche diode detectors gives an accurate characterization of gain-switched semiconductor lasers with picosecond resolution. The high sensitivity and the clean shape of the time response reveal even small features (reflections and relaxation oscillations), making a true optimization of the laser-diode operation possible. The technique outperforms the standard characterization with ultrafast p-i-n photodiodes and a sampling oscilloscope. In addition, compared with other methods, it has favorable features that greatly simplify the measurement.

A new single-photon avalanche diode (SPAD) with double-epitaxial silicon structure is presented. The device has a time response with short diffusion tail (270 ps time-constant), high resolution (45 ps FWHM, full-width at half maximum of the peak) and low noise, i. e. low dark-count rate.

A new silicon single-photon avalanche diode (SPAD) with epitaxial structure is presented. The carrier diffusion effect, which has plagued the time response of previous SPADs, is strongly reduced. The resolution obtained, less than 30ps full width at half-maximum, is the highest so far reported in single-photon timing.

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.

Over the past few years there has been a growing interest in monolithic arrays of single photon avalanche diodes (SPAD) for time resolved detection of faint ultrafast optical signals. SPADs implemented in CMOS-compatible planar technologies offer the typical advantages of microelectronic devices (small size, ruggedness, low voltage, low power, etc.). Furthermore, they have inherently higher photon detection efficiency than PMTs and are able to provide, beside sensitivities down to single-photons, very high acquisition speeds. They are in principle therefore ideal candidates for the development of new parallel systems analysis. The birth of novel techniques and diagnostic instruments in fact has led towards the parallelization of measurement systems and consequently to the development of monolithic arrays of detectors. Unfortunately, the implementation of a multidimensional system is a challenging task, because optical and electrical crosstalk between adjacent channels strongly affect the timing performances of the SPADs; for these reasons, only a few number of commercial solutions are available and their performances are not comparable to the best single channel ones. A new compact module based on a 8x1 high performance time resolved SPAD array with a new timing approach is here presented.