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Multi-dimensional time correlated single-photon counting has reached a prominent position among analytical techniques employed in the medical and biological fields. The development of instruments able to perform temporal and spectral fluorescence analysis (sFLIM) at the same time is limited by the performance of single-photon detectors, since currently available arrays cannot simultaneously satisfy 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 and low dark count rate, 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 end, 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 (>5 MHz) and low after-pulsing (<2%). Finally, the output timing signals are read and conditioned by proper CMOS electronics. The complete system shows a very good temporal resolution of about 45 ps (FWHM). [ABSTRACT FROM AUTHOR]

After a brief review of the physics of photon detection in single photon avalanche diode (SPAD) devices, in this paper we will outline the principle of operation of a model we developed with the aim of calculating both photon detection efficiency (PDE) and temporal response (TR) of these detectors. Then we will apply the model to the devices currently available in order to critically analyze some experimental results. We will show in particular how the use of the model allows us to gain a better understanding of the influence of each device parameter in determining both the PDE and the TR. Finally we will discuss some modifications that can be applied to the device structure in order to overcome such limitations. Their effectiveness in improving both the PDE and the TR will be investigated by means of the aforementioned model. The aim is to provide the reader with an insight of which performances can be expected in the next few years if a strong development of the SPAD structure is pursued. [ABSTRACT FROM AUTHOR]

Recent advances in fluorescence measurements for single molecule spectroscopy, genomics, proteomics and medical diagnostics require single-photon detectors with high quantum efficiency in the extended red spectral range (from 600 nm to 900 nm) and low noise (<50 kc s-1). Amongst industrial production, avalanche photodiodes with large diameter (up to 500 µm) are available which can work in Geiger mode (GM) with good photon detection efficiency (higher than 20%) and some of them also with quite low dark counting rate. They allow one to attain high collection efficiency with simple optical systems. However, they work at high bias voltage (over 200 V) with high power dissipation and are easily damaged by exposure to intense light. A detector carrier module has been designed for safe operation and full performance exploitation of any silicon device suitable for GM operation, with any breakdown voltage up to 480 V. Efficient photon counting and timing is achieved in a very compact module by means of an integrated active-quenching circuit (iAQC) with fast time pickup. Catastrophic failure is avoided by a dedicated monitor and safety circuit, even in the case of exposure to sunlight. [ABSTRACT FROM AUTHOR]

The evolution of solid-state avalanche detectors of single optical photons is outlined and the issues for further progress are discussed. Physical phenomena that underlay the operation of the single-photon avalanche diodes (SPAD) and determine the performance are considered and their role is assessed (detection efficiency; dark-counting rate; afterpulsing; photon timing resolution; etc.). The main technological issues that hamper the development of detectors with wide sensitive area and of array detectors with high filling factor are illustrated. Silicon SPADs are the main focus of attention; infrared-sensitive SPADs in germanium and in compound semiconductors are also dealt with. The role of the active-quenching circuits (AQC) is assessed and the evolution is outlined up to integrated AQCs, which offer the prospect of monolithic integration of complete photon counter instruments. [ABSTRACT FROM AUTHOR]

Malignant pleural mesothelioma usually arises from the pleural surface and progressively encases the lungs. Pulmonary involvement generally occurs at an advanced stage, while intraparenchymal nodules, in the absence of pleural lesions, constitute a less frequent presentation. We describe the case of a patient with multiple bilateral pulmonary nodules, mediastinal lymphadenopathies and left pleural effusion in the absence of pleural lesions, simulating advanced stage lung cancer. Thoracoscopic inspection did not detect any lesions. Pathological examination on one pulmonary nodule revealed malignant pleural mesothelioma. Despite its rarity, intraparenchymal malignant pleural mesothelioma should always be taken into account, when lung nodules are present, to prevent misdiagnosis and avoid delayed treatment.

Objectives: To describe sonographic features of the microcystic elongated and fragmented (MELF) pattern of myometrial invasion (MI) using the International Endometrial Tumor Analysis (IETA) criteria; to assess the effect of the MELF pattern on preoperative ultrasound evaluation of MI; and to determine the relationship of the MELF pattern to more advanced stage (≥ IB) and lymph node metastases in women with endometrioid endometrial cancer., Methods/materials: We included 850 women with endometrioid endometrial cancer from the prospective IETA 4 study. Ultrasound experts performed all ultrasound examinations, according to the IETA protocol. Reference pathologists assessed the presence or absence of the MELF pattern. Sonographic features and accuracy of ultrasound assessment of MI were compared in cases with the presence and the absence of the MELF pattern. The MELF pattern was correlated to more advanced stage (≥IB) and lymph node metastases., Results: The MELF pattern was present in 197 (23.2%) women. On preoperative ultrasound imaging the endometrium was thicker (p = 0.031), more richly vascularized (p = 0.003) with the multiple multifocal vessel pattern (p < 0.001) and the assessment of adenomyosis was more often uncertain (p < 0.001). The presence or the absence of the MELF pattern did not affect the accuracy of the assessment of MI. The MELF pattern was associated with deep myometrial invasion (≥ 50%) (p < 0.001), cervical stromal invasion (p = 0.037), more advanced stage (≥ IB) (p < 0.001) and lymph node metastases (p = 0.011)., Conclusions: Tumors with the MELF pattern were slightly larger, more richly vascularized with multiple multifocal vessels and assessment of adenomyosis was more uncertain on ultrasound imaging. The MELF pattern did not increase the risk of underestimating MI in preoperative ultrasound staging. Tumors with the MELF pattern were more than twice as likely to have more advanced stage (≥ IB) and lymph node metastases., (© IGCS and ESGO 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

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.

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.

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.

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 V oυ = 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., Competing Interests: Conflict of interest statement: M. Ghioni discloses equity in Micro Photon Devices S.r.l. (MPD).