Wide-field Fluorescence Lifetime Imaging Microscopy with a High-Speed Mega-pixel SPAD Camera

Abstract

Fluorescence lifetime imaging microscopy (FLIM) is a key technology that provides direct insight into cell metabolism, cell dynamics and protein activity. However, determining the lifetimes of different fluorescent proteins requires the detection of a relatively large number of photons, hence slowing down total acquisition times. Moreover, there are many cases, for example in studies of cell collectives, where wide-field imaging is desired. We report scan-less wide-field FLIM based on a 0.5 Megapixel resolution, time-gated Single Photon Avalanche Diode (SPAD) camera, with acquisition rates up to 1 Hz. Fluorescence lifetime estimation is performed via a pre-trained artificial neural network with 1000-fold improvement in processing times compared to standard least squares fitting techniques. We utilised our system to image HT1080 -human fibrosarcoma cell line as well as Convallaria. The results show promise for real-time FLIM and a viable route towards multi-megapixel fluorescence lifetime images, with a proof-of-principle mosaic image shown with 3.6 megapixels.

Imaging Dynamics Beneath Turbid Media via Parallelized Single‐Photon Detection

“…[21] Recently developed SPAD arrays, based on standard complementary metaloxide-semiconductor(CMOS) fabrication technology, can integrate up to a million SPAD pixels onto a small chip. [22,23] This led to new imaging applications in fluorescence lifetime imaging, [24] scanning microscopy, [25] confocal fluorescence fluctuation spectroscopy, [26] Fourier ptychography, [27] as well as com-puter vision tasks, such as depth profile estimation, [22,28] seeing around corners [29] and through scattering slabs. [4,30] Most prior DCS measurement systems relied on fast single-pixel singlephoton detectors (including single-pixel SPAD and photomultiplier tubes) for optical measurement.…”

Imaging dynamics beneath turbid media via parallelized single-photon detection

“…To solve the first two challenges listed above, we use a single-photon avalanche diode (SPAD) array to simultaneously measure speckle field fluctuations across the tissue surface at the requisite sampling rates (∼µs) and single-photon sensitivities needed for deep detection [19]. Recently developed SPAD arrays, based now on standard complementary metal-oxide-semiconductor(CMOS) fabrication technology, can integrate up to a million SPAD pixels onto a small chip [20,21] for new scientific imaging applications in fluorescence lifetime imaging [22], scanning microscopy [23], confocal fluorescence fluctuation spectroscopy [24], Fourier ptychography [25], as well as computer vision tasks such as depth profile estimation [20,26], seeing around corners [27] and through scattering slabs [28,4]. Most prior DCS measurement systems relied on fast single-pixel single-photon detectors (including SPADs and photomultiplier tubes) for optical measurement [16].…”

Quantum illumination imaging with a single-photon avalanche diode camera

“…Their implementation in standard CMOS technology [23] has triggered the development of digital SPAD-based cameras [24,25]. Thus far, these imaging devices have shown their capabilities in fluorescence lifetime imaging [26][27][28][29][30], LiDAR [31][32][33][34], non-line-of-sight imaging [35] and imaging through scattering media [36]. In quantum optics, a few experimental studies have used SPAD cameras for characterising spatial correlations [37] and entanglement [38,39] between entangled pairs.…”