RESUMO
The minimization of Single Photon Avalanche Diodes (SPADs) dead time is a key factor to speed up photon counting and timing measurements. We present a fully integrated Active Quenching Circuit (AQC) able to provide a count rate as high as 100 MHz with custom technology SPAD detectors. The AQC can also operate the new red enhanced SPAD and provide the timing information with a timing jitter Full Width at Half Maximum (FWHM) as low as 160 ps.
RESUMO
We propose a novel circuit for single photon avalanche diode (SPAD) current read-out, for photon timing applications. The circuit consists of a single transistor trans-impedance amplifier with a GHz bandwidth: the feedback loop fixes the SPAD anode voltage and allows us to obtain a high time resolution with a very high equivalent current threshold (almost 700 µA). The trans-impedance stage is followed by a low pass filter that reduces the crosstalk of other on-chip detectors and makes the designed structure suitable for multi-detector systems. The discrete components prototype presented in this letter achieves a state-of-art resolution of 34.4 ps FWHM, presents negligible crosstalk between the different pixels and opens the way for the development of an integrated structure with a large number of channels.
RESUMO
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.
Assuntos
Elétrons , Microscopia de Fluorescência/métodos , Imagem Molecular/instrumentação , Fótons , Biologia Computacional , Difusão , Desenho de Equipamento , Fluorescência , Conformação Molecular , Imagem Molecular/métodos , Sensibilidade e Especificidade , Fatores de TempoRESUMO
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).
Assuntos
Equipamentos e Provisões Elétricas , Fótons , Capacitância Elétrica , Probabilidade , Fatores de Tempo , Transistores EletrônicosRESUMO
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.
RESUMO
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.
RESUMO
Solution-based single-molecule fluorescence spectroscopy is a powerful new experimental approach with applications in all fields of natural sciences. The basic concept of this technique 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 obtained with different novel multipixel single-photon counting detectors.
RESUMO
We present a new photon timing circuit that achieves a time resolution of 35 ps full width at half maximum with single photon avalanche diodes having active area diameters up to 200 microm. The timing circuit is based on a double avalanche current sensing network that makes it particularly suited to operation at high photon counting rates. Thanks to its self-adjusting capabilities, no trimming is needed even when changing the photodetector operating conditions over a wide range.