SPAD-based asynchronous-readout array detectors for image-scanning microscopy

Abstract

Fluorescence microscopy and derived techniques are continuously looking for photodetectors able to guarantee increased sensitivity, high spatial and temporal resolution and ease of integration into modern microscopy architectures. Recent advances in single-photon avalanche diodes (SPADs) fabricated with industry-standard microelectronic processes allow the development of new detection systems tailored to address the requirements of advanced imaging techniques (such as image-scanning microscopy). To this aim, we present the complete design and characterization of two bidimensional SPAD arrays composed of 25 fully independent and asynchronously-operated pixels, both having fill-factor of about 50% and specifically designed for being integrated into existing laser scanning microscopes. We used two different microelectronics technologies to fabricate our detectors: the first technology exhibiting very low noise (roughly 200 dark counts per second at room temperature), and the second one showing enhanced detection efficiency (more than 60% at a wavelength of 500 nm). Starting from the silicon-level device structures and moving towards the in-pixel and readout electronics description, we present performance assessments and comparisons between the two detectors. Images of a biological sample acquired after their integration into our custom imagescanning microscope finally demonstrate their exquisite on-field performance in terms of spatial resolution and contrast enhancement. We envisage that this work can trigger the development of a new class of SPAD-based detector arrays able to substitute the typical singleelement sensor used in fluorescence laser scanning microscopy.

Confocal-based fluorescence fluctuation spectroscopy with a SPAD array detector

“…The current generation of 0.35 µm high-voltage complementary metal-oxide-semiconductor (HVCMOS) SPAD detectors suffers from after-pulsing, but we have shown several methods to compensate for this effect. It is worth noting that other types of SPAD technology fabrication, such as 0.16 µm bipolar-CMOS-DMOS (BCD) 22 , have an after-pulsing probability far below 1% at the same hold-off time—more than one order of magnitude less than the HVCMOS technology used here. On the other hand, the dark count rate of BCD SPAD array detectors can be significantly higher than that of their HVCMOS counterparts, but since the dark counts were uncorrelated, they would not severely affect the FCS autocorrelation curves.…”

“…The measurements were performed with a 5×5 silicon SPAD array detector fabricated using 0.35 µm HVCMOS technology 22 . Compared to other larger SPAD detectors 37 , 39 , this detector has a much higher fill factor (approximately 50%) and is therefore better suited for imaging the detection volume.…”

“…Recently, our group introduced a new class of asynchronous-readout silicon SPAD array detectors 22 with temporal and spatial characteristics that can overcome these limitations. Similar to the AiryScan detector, these SPAD array detectors allow imaging of the fluorescence detection volume but with a practically unlimited frame rate, and they temporally tag the fluorescence photons with picosecond precision; see Fig.…”

Optical nanoscopy

“…When spinning the disk at a fast rate, the sample undergoes excitation several hundred times per second. The emitted light is typically collected and imaged by a high-resolution and high quantum efficiency CCD camera or an array of sensors like modern SPAD array devices [76]. One meaningful advantage in the approach with respect to the previous one is an improvement of temporal resolution without compromising the spatial one.…”

Two-photon image-scanning microscopy with SPAD array and blind image reconstruction

“…In this context it is important to highlight that the parallel nature of the SPAD array detector increases the imaging dynamic range [63]. Despite the relatively long hold-off/dead time of each single SPAD array element (200 ns in the device used here, 20 ns in more recent models [60]), because the fluorescent photons are spread across the many elements of the SPAD array, its effective dynamic range is rather high. In fact we have already used the SPAD array with resonant scanners, as was shown in [17].…”