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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).

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.