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.

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.

Prospective trial evaluating new circular and linear stapler devices for gastrointestinal anastomosis: preliminary data.

Several commercial models of stapler devices are available. This study evaluated the ease of use, effectiveness and safety of new commercial stapling devices for gastrointestinal anastomosis. A total of 11 patients (5 men) requiring surgical therapy for benign or malignant disease of the digestive tract were recruited between July and October 2006. Eleven patients were treated with KYGW circular stapler or KYFB linear stapler (Changzhou Kangdi Medical Stapler). In these patients, 14 staplers were used and 21 stapled sutures (16 linear, 5 circular) were performed. Number of anastomoses successfully completed, postoperative anastomotic fistula or dehiscence, days to take fluid and normal diet, length of hospital stay and anastomotic stenosis were recorded. A 10-point questionnaire enquiring about the instrument and anastomotic features was administered to surgeons immediately after the operation in the study group and in 10 control patients treated with standard CDH circular and SDH linear staples (Ethicon Endo-Surgery). Mean scores on the questionnaire for the experimental and control groups were good (>7.5) and did not significantly differ for handling, closing ease, bleeding, and overall satisfaction. No case of intra-abdominal sepsis, leakage or intestinal obstruction was recorded in the study group. In the 5 patients with colorectal anastomosis, the anastomotic lumen at 15 days was wide open and at 3 months there were no strictures. These new instruments are valuable for performing gastrointestinal anastomosis and are in conformity with clinical requirements; their use is simple and seems to be safe.