In this paper, we describe a novel solution to increase the speed of Time-Correlated Single Photon Counting (TCSPC) measurements by almost an order of magnitude while providing, in principle, zero distortion regardless of the experimental conditions. Typically, the relatively long dead time associated with the conversion electronics requires a proper tune of the excitation power in order to avoid distortions of the reconstructed waveform due to pileup and counting loss. As a result, the maximum operating rate of a TCSPC channel is now limited between 1% and 5% of the excitation frequency, thus leading to relatively long acquisition times. We show that negligible distortion (below 1%) is guaranteed if the dead time associated with the converter is kept below the dead time of the detector, and at the same time the detector dead time is matched to the duration of the excitation period. In this way, unprecedented high-speed operation is possible. In this paper, we provide a theoretical analysis of the technique, including the main non-idealities which are introduced by a generic physical implementation. The results are supported by both numerical simulations and analytical calculations.

Time-Correlated Single Photon Counting (TCSPC) has been long recognized as the most sensitive method for fluorescence lifetime measurements, but often requiring "long" data acquisition times. This drawback is related to the limited counting capability of the TCSPC technique, due to pile-up and counting loss effects. In recent years, multi-module TCSPC systems have been introduced to overcome this issue. Splitting the light into several detectors connected to independent TCSPC modules proportionally increases the counting capability. Of course, multi-module operation also increases the system cost and can cause space and power supply problems. In this paper, we propose an alternative approach based on a new detector and processing electronics designed to reduce the overall system dead time, thus enabling efficient photon collection at high excitation rate. We present a fast active quenching circuit for single-photon avalanche diodes which features a minimum dead time of 12.4 ns. We also introduce a new Time-to-Amplitude Converter (TAC) able to attain extra-short dead time thanks to the combination of a scalable array of monolithically integrated TACs and a sequential router. The fast TAC (F-TAC) makes it possible to operate the system towards the upper limit of detector count rate capability (∼80 Mcps) with reduced pile-up losses, addressing one of the historic criticisms of TCSPC. Preliminary measurements on the F-TAC are presented and discussed. C 2015 AIP Publishing LLC. [http://dx

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.

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Current instruments used to detect specific protein-protein interactions in live cells for applications in high-content screening (HCS) are limited by the time required to measure the lifetime. Here, a 32 × 1 single-photon avalanche diode (SPAD) array was explored as a detector for fluorescence lifetime imaging (FLIM) in HCS. Device parameters and characterization results were interpreted in the context of the application to determine if the SPAD array could satisfy the requirements of HCS-FLIM. Fluorescence lifetime measurements were performed using a known fluorescence standard; and the recovered fluorescence lifetime matched literature reported values. The design of a theoretical 32 × 32 SPAD array was also considered as a detector for a multi-point confocal scanning microscope.