After three decades of developments, single particle tracking (SPT) has become a powerful tool to interrogate dynamics in a range of materials including live cells and novel catalytic supports because of its ability to reveal dynamics in the structure–function relationships underlying the heterogeneous nature of such systems In this review, we summarize the algorithms behind, and practical applications of, SPT We first cover the theoretical background including particle identification, localization, and trajectory reconstruction General instrumentation and recent developments to achieve two- and three-dimensional subdiffraction localization and SPT are discussed We then highlight some applications of SPT to study various biological and synthetic materials systems Finally, we provide our perspective regarding several directions for future advancements in the theory and application of SPT

Single Particle Tracking: From Theory to Biophysical Applications

Single-photon avalanche diode (SPAD) arrays are solid-state detectors that offer imaging capabilities at the level of individual photons, with unparalleled photon counting and time-resolved performance. This fascinating technology has progressed at a very fast pace in the past 15 years, since its inception in standard CMOS technology in 2003. A host of architectures have been investigated, ranging from simpler implementations, based solely on off-chip data processing, to progressively "smarter" sensors including on-chip, or even pixel level, time-stamping and processing capabilities. As the technology has matured, a range of biophotonics applications have been explored, including (endoscopic) FLIM, (multibeam multiphoton) FLIM-FRET, SPIM-FCS, super-resolution microscopy, time-resolved Raman spectroscopy, NIROT and PET. We will review some representative sensors and their corresponding applications, including the most relevant challenges faced by chip designers and end-users. Finally, we will provide an outlook on the future of this fascinating technology.

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Single-photon avalanche diode imagers in biophotonics: review and outlook

We present a CMOS imager consisting of 32×32 smart pixels, each one able to detect single photons in the 300-900 nm wavelength range and to perform both photon-counting and photon-timing operations on very fast optical events with faint intensities. In photon-counting mode, the imager provides photon-number (i.e, intensity) resolved movies of the scene under observation, up to 100 000 frames/s. In photon-timing, the imager provides photon arrival times with 312 ps resolution. The result are videos with either time-resolved (e.g., fluorescence) maps of a sample, or 3-D depth-resolved maps of a target scene. The imager is fabricated in a cost-effective 0.35-μm CMOS technology, automotive certified. Each pixel consists of a single-photon avalanche diode with 30 μm photoactive diameter, coupled to an in-pixel 10-bit time-to-digital converter with 320-ns full-scale range, an INL of 10% LSB and a DNL of 2% LSB. The chip operates in global shutter mode, with full frame times down to 10 μs and just 1-ns conversion time. The reconfigurable imager design enables a broad set of applications, like time-resolved spectroscopy, fluorescence lifetime imaging, diffusive optical tomography, molecular imaging, time-of-flight 3-D ranging and atmospheric layer sensing through LIDAR.

CMOS Imager With 1024 SPADs and TDCs for Single-Photon Timing and 3-D Time-of-Flight

Fluorescence lifetime imaging (FLIM): Basic concepts and some recent developments

We report on the design and characterization of a multipurpose 64 × 32 CMOS single-photon avalanche diode (SPAD) array. The chip is fabricated in a high-voltage 0.35-μm CMOS technology and consists of 2048 pixels, each combining a very low noise (100 cps at 5-V excess bias) 30-μm SPAD, a prompt avalanche sensing circuit, and digital processing electronics. The array not only delivers two-dimensional intensity information through photon counting in either free-running (down to 10-μs integration time) or time-gated mode, but can also perform smart light demodulation with in-pixel background suppression. The latter feature enables phase-resolved imaging for extracting either three-dimensional depth-resolved images or decay lifetime maps, by measuring the phase shift between a modulated excitation light and the reflected photons. Pixel-level memories enable fully parallel processing and global-shutter readout, preventing motion artifacts (e.g., skew, wobble, motion blur) and partial exposure effects. The array is able to acquire very fast optical events at high frame-rate (up to 100 000 fps) and at single-photon level. Low-noise SPADs ensure high dynamic range (up to 110 dB at 100 fps) with peak photon detection efficiency of almost 50% at 410 nm. The SPAD imager provides different operating modes, thus, enabling both time-domain applications, like fluorescence lifetime imaging (FLIM) and fluorescence correlation spectroscopy, as well as frequency-domain FLIM and lock-in 3-D ranging for automotive vision and lidar.

100 000 Frames/s 64 × 32 Single-Photon Detector Array for 2-D Imaging and 3-D Ranging

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.

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O. H. W. Siegmund

Papers

Development of new photon-counting detectors for single-molecule fluorescence microscopy