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Imaging techniques based on time-correlated single photon counting (TCSPC), such as fluorescence lifetime imaging microscopy (FLIM), rely on fast single-photon detectors as well as timing electronics in the form of time-to-digital or time-to-analog converters. Conventional systems rely on stand-alone or small arrays (up to 32) of detectors and exte...
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In this review, we discuss methods and advancements in fluorescence lifetime imaging microscopy that permit measurements to be performed at faster speed and higher resolution than previously possible. We review fast single-photon timing technologies and the use of parallelized detection schemes to enable high-throughput and high content imaging app...
Time-correlated single-photon counting (TCSPC) has been the gold standard for fluorescence lifetime imaging (FLIM) techniques due to its high signal-to-noize ratio and high temporal resolution. The sensor system's temporal instrument response function (IRF) should be considered in the deconvolution procedure to extract the real fluorescence decay t...
Fluorescence lifetime imaging (FLIM) is widely applied to obtain quantitative information from fluorescence signals, particularly using Förster Resonant Energy Transfer (FRET) measurements to map, for example, protein-protein interactions. Extracting FRET efficiencies or population fractions typically entails fitting data to complex fluorescence de...
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Citations
... Current single-photon avalanche diode (SPAD) cameras, based on photon counting, already achieve resolutions in the megapixel regime. However, for many applications, such as ToF-LiDAR [1][2][3][4][5][6], fluorescence lifetime imaging (FLIM) [7][8][9][10], biophotonics [11][12][13][14], and quantum sensing [15,16]. ...
Single-photon detection and timing has attracted increasing interest in recent years due to their necessity in the field of quantum sensing and the advantages of single-quanta detection in the field of low-level light imaging. While simple bucket detectors are mature enough for commercial applications, more complex imaging detectors are still a field of research comprising mostly prototype-level detectors. A major problem in these detectors is the implementation of in-pixel timing circuitry, especially for two-dimensional imagers. One of the most promising approaches is the use of voltage-controlled ring resonators in every pixel. Each of these runs independently based on a voltage supplied by a global reference. However, this yields the problem that the supply voltage can change across the chip which, in turn, changes the period of the ring resonator. Due to additional parasitic effects, this problem can worsen with increasing measurement time, leading to drift in the timing information. We present here a method to identify and correct such temporal drifts in single-photon detectors based on asynchronous quantum ghost imaging. We also show the effect of this correction on recent quantum ghost imaging (QGI) measurement from our group.
... We used various wavelengths of picosecond lasers to examine the different electric field distributions at various depths of the device. The device exhibited the largest jitter value when subjected to the 405nm wavelength, commonly used in FLIM applications [43,44], and the 905nm wavelength, commonly used in automotive Lidar applications [45,46], also showed a significantly larger jitter value compared to the 780nm wavelength, commonly used in pulsed Lidar imaging and high spectral resolution Lidar applications [47,48,49], as shown in Figure 7(b). ...
We have proposed a structural design for a single photon avalanche diode with a low breakdown voltage. This diode is fabricated using Taiwan Semiconductor Manufacturing Company (TSMC) 0.18 μm HV CMOS technology, and it can maintain a high operating excess voltage in an n-on-p design without requiring any additional customized well layers. The n-on-p type device is particularly advantageous for a 3D-stacked backside illuminated structure and offers excellent photon detection capabilities at longer wavelengths. By incorporating a high doping concentration PDD well layer, we can significantly increase the excess bias, resulting in enhanced photon detection probability in the near-infrared wavelength range, all while maintaining a lower voltage due to a reduction in breakdown voltage. This design also leads to power consumption savings. As a result, our designed device is well-suited for consumer applications such as 3D image rendering and Lidar technology.
... However, most fast cameras do not exhibit pixelbased fluorescence lifetime capabilities. Single-photon avalanche diode (SPAD)-based arrays are now emerging, where each pixel can have lifetime capabilities[55][56][57][58] . We envision the next generation of FRETsael would be in the context of a wide-field microscope with lifetime-based SPAD arrays acting as lifetime-capable cameras, which will allow nm localizations of biomolecular interactions in live cells in as close as it can get to the biological equivalent of real-time. ...
Super-resolution light microscopy techniques facilitate the observation of nanometer-size biomolecules, which are 1-2 orders of magnitude smaller than the diffraction limit of light. Using super-resolution microscopy techniques it is possible to observe fluorescence from two biomolecules in close proximity, however not necessarily in direct interaction. Using FRET-sensitized acceptor emission localization (FRETsael), we localize biomolecular interactions exhibiting FRET with nanometer accuracy, from two color fluorescence lifetime imaging data. The concepts of FRETsael were tested first against simulations, in which the recovered localization accuracy is 20-30 nm for true-positive detections of FRET pairs. Further analyses of the simulation results report the conditions in which true-positive rates are maximal. We then show the capabilities of FRETsael on simulated samples of Actin-Vinculin and ER-ribosomes interactions, as well as on experimental samples of Actin-Myosin two-color confocal imaging. Conclusively, the FRETsael approach paves the way towards studying biomolecular interactions with improved spatial resolution from laser scanning confocal two color fluorescence lifetime imaging.
... However, they usually utilize vertical p-i-n structures, which are less favorable for the planar CMOS process. Instead, planar PD structures are of particular interest due to their ability for integration with electronics in planar CMOS processes, and thus suitable for EPICs [27]. In addition, for planar PDs, the decoupling of the photon absorption length and carrier collection path results in no trade-off between the 3-dB bandwidth and quantum efficiency. ...
We report normal-incidence planar GeSn resonant-cavity-enhanced photodetectors (RCE-PDs) with a lateral $p \text{-} i \text{-} n$ p - i - n homojunction configuration on a silicon-on-insulator (SOI) platform for short-wave infrared (SWIR) integrated photonics. The buried oxide of the SOI platform and the deposited ${\rm{Si}}{{\rm{O}}_2}$ S i O 2 layer serve as the bottom and top reflectors, respectively, creating a vertical cavity for enhancing the optical responsivity. The planar $p \text{-} i \text{-} n$ p - i - n diode structure is favorable for complementary-metal-oxide-semiconductor-compatible, large-scale integration. With the bandgap reduction enabled by the 4.2% Sn incorporation into the GeSn active layer, the photodetection range extends to 1960 nm. The promising results demonstrate that the developed planar GeSn RCE-PDs are potential candidates for SWIR integrated photonics.
... One of the most relevant characteristics in threedimensional (3D) imaging and ranging applications based on Single-Photon Avalanche Diode (SPAD) is the direct Time-of-Flight (ToF) capability [1]. To this end, high-resolution TDCs are highly demanded for 3D imaging [2,3], Fluorescence Lifetime Imaging Microscopy (FLIM) [4,5], and Positron Emission Tomography (PET) [6,7]. Furthermore, the increase in the number of detector modules and the requirement for real-time acquisition have led to the widespread utilization of multi-channel TDCs. ...
In this paper, we present a proposed field programmable gate array (FPGA)-based time-to-digital converter (TDC) architecture to achieve high performance with low usage of resources. This TDC can be employed for multi-channel direct Time-of-Flight (ToF) applications. The proposed architecture consists of a synchronizing input stage, a tuned tapped delay line (TDL), a combinatory encoder of ones and zeros counters, and an online calibration stage. The experimental results of the TDC in an Artix-7 FPGA show a differential non-linearity (DNL) in the range of [−0.953, 1.185] LSB, and an integral non-linearity (INL) within [−2.750, 1.238] LSB. The measured LSB size and precision are 22.2 ps and 26.04 ps, respectively. Moreover, the proposed architecture requires low FPGA resources.
... Single-photon avalanche diodes (SPADs), due to their single photon sensitivity, have been employed in many photon-starved applications such as fluorescence lifetime imaging (FLI), time-of-flight (TOF) and conventional imaging [1][2][3][4][5][6][7]. The possibility of fabricating SPADs in a conventional CMOS process facilitates mass production with lower associated costs. ...
Single-photon avalanche diodes (SPADs) fabricated in conventional CMOS processes typically have limited near infra-red (NIR) sensitivity. This is the consequence of isolating the SPADs in a lowly-doped deep N-type well. In this work, we present a second improved version of the “current-assisted” single-photon avalanche diode, fabricated in a conventional 350 nm CMOS process, having good NIR sensitivity owing to 14 μm thick epilayer for photon absorption. The presented device has a photon absorption area of 30 × 30 µm2, with a much smaller central active area for avalanche multiplication. The photo-electrons generated in the absorption area are guided swiftly towards the central area with a drift field created by the “current-assistance” principle. The central active avalanche area has a cylindrical p-n junction as opposed to the square geometry from the previous iteration. The presented device shows improved performance in all aspects, most notably in photon detection probability. The p-n junction capacitance is estimated to be ~1 fF and on-chip passive quenching with source followers is employed to conserve the small capacitance for bringing monitoring signals off-chip. Device physics simulations are presented along with measured dark count rate (DCR), timing jitter, after-pulsing probability (APP) and photon detection probability (PDP). The presented device has a peak PDP of 22.2% at a wavelength of 600 nm and a timing jitter of 220 ps at a wavelength of 750 nm.
... Alternatively, widefield FLIM systems acquire spatial data in parallel. Nonetheless, to achieve high temporal resolution, they still need to temporally scan either a gated window 25,26 or detection phase 27 , or they must use TCSPC which requires a large number of repetitive measurements to construct a temporal histogram 28,29 . Limited by the scanning requirement, current FLIM systems operate at only a few frames per second when acquiring high-resolution images 21,30,31 . ...
We present high-resolution, high-speed fluorescence lifetime imaging microscopy (FLIM) of live cells based on a compressed sensing scheme. By leveraging the compressibility of biological scenes in a specific domain, we simultaneously record the time-lapse fluorescence decay upon pulsed laser excitation within a large field of view. The resultant system, referred to as compressed FLIM, can acquire a widefield fluorescence lifetime image within a single camera exposure, eliminating the motion artifact and minimizing the photobleaching and phototoxicity. The imaging speed, limited only by the readout speed of the camera, is up to 100 Hz. We demonstrated the utility of compressed FLIM in imaging various transient dynamics at the microscopic scale.
... Numerous strategies are in development for more sensitive detection methods. [130][131][132][133][134] 2.1.1 Wide-field FLIM WFI uses a parallel illumination field at the focus of the objective lens and collects fluorescence from the focal plane onto a camera (Fig. 5). ...
Significance:
Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique to distinguish the unique molecular environment of fluorophores. FLIM measures the time a fluorophore remains in an excited state before emitting a photon, and detects molecular variations of fluorophores that are not apparent with spectral techniques alone. FLIM is sensitive to multiple biomedical processes including disease progression and drug efficacy.
Aim:
We provide an overview of FLIM principles, instrumentation, and analysis while highlighting the latest developments and biological applications.
Approach:
This review covers FLIM principles and theory, including advantages over intensity-based fluorescence measurements. Fundamentals of FLIM instrumentation in time- and frequency-domains are summarized, along with recent developments. Image segmentation and analysis strategies that quantify spatial and molecular features of cellular heterogeneity are reviewed. Finally, representative applications are provided including high-resolution FLIM of cell- and organelle-level molecular changes, use of exogenous and endogenous fluorophores, and imaging protein-protein interactions with Förster resonance energy transfer (FRET). Advantages and limitations of FLIM are also discussed.
Conclusions:
FLIM is advantageous for probing molecular environments of fluorophores to inform on fluorophore behavior that cannot be elucidated with intensity measurements alone. Development of FLIM technologies, analysis, and applications will further advance biological research and clinical assessments.
... The choice between FLIM and seFRET often depends on the availability of specialist instrumentation (e.g., for TCSPC) or requirements such as fast acquisition speed (typically better for seFRET). However, breakthroughs in FLIM-enabling technologies [42][43][44][45][46][47][48] and data analysis [49][50][51] are reducing the barrier to adoption for FLIM; as the choice between FLIM and seFRET might slowly drift away from technical constraints, we aimed to develop a comparative analysis of their limits from an information theory perspective. ...
Förster resonance energy transfer (FRET) imaging is an essential analytical method in biomedical research. The limited photon-budget experimentally available, however, imposes compromises between spatiotemporal and biochemical resolutions, photodamage and phototoxicity. The study of photon-statistics in biochemical imaging is thus important in guiding the efficient design of instrumentation and assays. Here, we show a comparative analysis of photon-statistics in FRET imaging demonstrating how the precision of FRET imaging varies vastly with imaging parameters. Therefore, we provide analytical and numerical tools for assay optimization. Fluorescence lifetime imaging microscopy (FLIM) is a very robust technique with excellent photon-efficiencies. However, we show that also intensity-based FRET imaging can reach high precision by utilizing information from both donor and acceptor fluorophores.
... SPAD array sensors have single photon sensitivity, picosecond time resolution, and each pixel can independently perform TCSPC. While being a very promising technology due to excellent time resolution, current SPAD imagers still require improvements of the SPAD photon detection efficiency, array density and dark count rate [20,25]. ...
The properties of a novel ultra-fast optical imager, Tpx3Cam, were investigated for macroscopic wide-field phosphorescent lifetime imaging (PLIM) applications. The camera is based on a novel optical sensor and Timepix3 readout chip with a time resolution of 1.6 ns, recording of photon arrival time and time over threshold for each pixel, and readout rate of 80 megapixels per second. In this study, we coupled the camera to an image intensifier, a 760 nm emission filter and a 50 mm lens, and with a super-bright 627nm LED providing pulsed excitation of a 18 × 18 mm sample area. The resulting macro-imager with compact and rigid optical alignment of its main components was characterised using planar phosphorescent O2 sensors and a resolution plate mask. Several acquisition and image processing algorithms were evaluated to optimise the system resolution and performance for the wide-field PLIM, followed by imaging a variety of phosphorescent samples. The new PLIM system looks promising, particularly for phosphorescence lifetime-based imaging of O2 in various chemical and biological samples.
