Cooled SPAD array detector for low light-dose fluorescence laser scanning microscopy

Abstract

The single-photon timing and sensitivity performance and the imaging ability of asynchronous-readout single-photon avalanche diode (SPAD) array detectors have opened up enormous perspectives in fluorescence (lifetime) laser scanning microscopy (FLSM), such as super-resolution image scanning microscopy and high-information content fluorescence fluctuation spectroscopy. However, the strengths of these FLSM techniques depend on the many different characteristics of the detector, such as dark noise, photon-detection efficiency, after-pulsing probability, and optical cross talk, whose overall optimization is typically a trade-off between these characteristics. To mitigate this trade-off, we present, to our knowledge, a novel SPAD array detector with an active cooling system that substantially reduces the dark noise without significantly deteriorating any other detector characteristics. In particular, we show that lowering the temperature of the sensor to −15°C significantly improves the signal/noise ratio due to a 10-fold decrease in the dark count rate compared with room temperature. As a result, for imaging, the laser power can be decreased by more than a factor of three, which is particularly beneficial for live-cell super-resolution imaging, as demonstrated in fixed and living cells expressing green-fluorescent-protein-tagged proteins. For fluorescence fluctuation spectroscopy, together with the benefit of the reduced laser power, we show that cooling the detector is necessary to remove artifacts in the correlation function, such as spurious negative correlations observed in the hot elements of the detector, i.e., elements for which dark noise is substantially higher than the median value. Overall, this detector represents a further step toward the integration of SPAD array detectors in any FLSM system.

Single-photon microscopy to study biomolecular condensates

“…2 b). The measured diffusion coefficient 17.7 ± 0.3 μm 2 s −1 and the calculated diameter ( d = 24 nm) correspond well with previous measurements 5 , 6 , 27 and the manufacturer’s information. We further validate our svFCS approach on a more complex system where mobility is not only restricted to free diffusion.…”

“…In laser-scanning microscopy (LSM), FCS relies on confocality or two-photon excitation to generate the small detection volume (or probed region) and employs a fast single-point detector, such as a photomultiplier (PMT) or a single-photon avalanche diode (SPAD), to sample the fluorescence signal. However, recent advancements have demonstrated that the information content of LSM-based FCS increases by replacing the conventional single-point detector with a fast (≤μs) array detector capable of directly image the probed region 4 – 6 . Unlike single-point detectors, which average out the spatial variations in the fluorescence signal from the probed region, fast array detectors preserve spatial information without compromising temporal sampling.…”

Compact and effective photon-resolved image scanning microscope

“…Asynchronous read-out single-photon avalanche diode (SPAD) array detectors 1 – 3 are expected to terrifically extend the abilities of fluorescence laser-scanning microscopy (LSM). In contrast with the typical LSM single-element detectors, but similar to scientific cameras, this class of sensors preserves the spatial distribution of the impinging fluorescence photons.…”

“…10 Importantly, the same benefits have been easily transported to two-photon-excitation 16 and stimulated-emission depletion (STED) microscopy. 10 Fluorescence-fluctuation spectroscopy 17 (FFS) is another technique that has benefited from structured detection, 3,8 gaining access to molecular mobility information previously hidden. The aforementioned techniques require access to temporal scales of the order of the pixel dwell time or (histogram) bin time width for imaging or FFS, respectively.…”

Direct access to optical aberration information in fluorescence laser scanning microscopy using detector arrays

“…Presently, the most powerful detector array for implementing ISM in laser-scanning microscopy is represented by asynchronous read-out single-photon-avalanche diode (SPAD) array detectors. 5,6 The single-photon timing capability of these detectors has also facilitated their integration with time-resolved spectroscopy, resulting in further advancements in ISM and laser-scanning microscopy in general. In particular, these detector arrays have proven advantageous in enhancing resolution and contrast for imaging, [7][8][9][10] and improving information content and flexibility in fluorescence correlation spectroscopy.…”