KEYWORDS: Luminescence, Anisotropy, Image sensors, Biological research, Monte Carlo methods, Time correlated photon counting, Microscopy, Data acquisition, Virtual reality, Time metrology
A 128x192 SPAD array (QuantiCam) with an on-chip time-to-digital converter in each pixel is used as a camera in a single-photon time-resolved fluorescence microscope. The SPAD array introduces systematic nonlinearities and timing offset to the measured photon arrival times. This limits the fidelity of the experimental results. A Monte-Carlo algorithm was developed to transform the raw photon time-stamp stream coming from the SPAD array into a corrected virtual “photon” time-stamp stream devoid of the systematic measurement errors. This data is compatible with existing downstream data processing pipelines used in time-correlated single-photon counting. We discuss the calibration measurement, the algorithm, their performance and application to live fluorescence lifetime imaging of photosynthetic organisms.
We report the development of a novel massively-parallelised high-speed multifocal FLIM platform with the ability to acquire data 1024 times faster than a conventional TCSPC system. We demonstrate the system performanceFRET imaging of the fluorescent protein biosensor PercivalHR in iPSC derived neurons to measure the dynamic concentration of ADP/ATP in live cells. The advantages and performance envelope of the system will be shown and the potential for further applications explored.
Quantum 2.0 applications such as gravitational sensing require narrow linewidth lasers at specific wavelength and significant optical power. For single photon lidar applications such as to image through scattering media, intense yet short optical pulses are required. These are requirements not readily provided by existing laser systems. We suggest the use of master oscillator power amplifier (MOPA) systems consisting of a seed source providing the required spectral and temporal optical characteristics combined with a semiconductor based tapered amplifier to amplify the seed power to levels adequate for the required quantum applications. Considerations of the construction of such systems are discussed. Furthermore, there operational specifications will be determined and the suitability for quantum applications will be discussed.
Time-domain microfluidic fluorescence lifetime flow cytometry enables observation of fluorescence decay of particles or cells over time using time-correlated single photon counting (TCSPC). This method requires the fluorescence lifetime measured from a limited number of photons and in a short amount of time. In current implementations of the technique, the low throughput of state of the art detectors and lack of real-time statistical analysis of the current technology, the timedomain approaches are usually coupled with off-line analysis which impedes its use in flow cell sorting, tracking and capturing. In this work, we apply a 32×32 CMOS SPAD array (MegaFrame camera) for real-time imaging flow cytometry analysis. This technology is integrated into a 1024-beam multifocal fluorescence microscope and incorporating a microfluidic chip at the sample plane enables imaging of cell flow and identification. Furthermore, the 1.5% native pixel fill-factor of the MegaFrame camera is overcome using beamlet reprojection with <10 μW laser power at 490 nm for each beam. Novel hardware algorithms incorporating the center-of-mass method (CMM) with real-time background subtraction and division are implemented within the firmware, allowing lossless recording of TCSPC events at a 500 kHz frame rate with 1024 histogram bins at 52 ps time resolution. Live calculation of background compensated CMM-based fluorescence lifetime is realized at a user-defined frame rate (typically 0.001 ~ 27 kHz) for each SPAD pixel. The work in this paper considers the application of the SPAD array to confocal fluorescence lifetime imaging of multiple coincident particles flowing within a microfluidic channel. Compared to previous flow systems based on single-point detectors, the multi-beam flow system enables visualization, detection and categorization of multiple groups of cells or particles according to their fluorescence lifetime.
Single-Photon Avalanche Diode (SPAD) sensors are one of the detectors of choice in LIDAR applications, due to their high sensitivity and time resolution. Traditionally, single-point SPAD detectors have been used, necessitating optical scanning, and hence leading to slower acquisition times. However, recent advances in SPAD technology have yielded high pixel count imagers. In these sensors, high sensitivity is ensured by maximising the photo-sensitive area of the device, whilst time-resolved capability is offered by the time stamping of photon detections and/or a programmable timegate synced to the laser source. The resulting sensors show an exciting promise for LIDAR, especially in challenging, low-light, high-speed applications.
A photonic lantern is an adiabatic guided-wave transition between a multimode waveguide and a set of single-mode cores. As such, photonic lanterns facilitate the efficient coupling of multimode light to single-mode devices, examples of which include fibre Bragg gratings and arrayed waveguide gratings. In this work, we demonstrate that photonic lanterns based on tapered multicore fibres (MCFs) provide a potentially powerful new route to efficiently couple multimode states of light to a two-dimensional array of Single Photon Avalanche Detectors (SPADs). The SPAD array consists of a 32×32 square array of pixels, each of which has its own time to digital converter (TDC) for Time Correlated Single Photon Counting (TCSPC) with a timing resolution of 55 ps. For our application, the geometry of the MCF used to fabricate the photonic lantern was chosen such that each single mode in the MCF can be mapped onto an individual SPAD pixel. Upon injecting a broad supercontinuum signal into a 290 m long MCF via a photonic lantern, wavelength-to-time mapped spectra were obtained from all modes. We believe that the techniques we report here may find applications in areas such as Raman spectroscopy, coherent LIDAR, and quantum optics.
The SPADnet FP7 European project is aimed at a new generation of fully digital, scalable and networked photonic components to enable large area image sensors, with primary target gamma-ray and coincidence detection in (Time-of- Flight) Positron Emission Tomography (PET). SPADnet relies on standard CMOS technology, therefore allowing for MRI compatibility. SPADnet innovates in several areas of PET systems, from optical coupling to single-photon sensor architectures, from intelligent ring networks to reconstruction algorithms. It is built around a natively digital, intelligent SPAD (Single-Photon Avalanche Diode)-based sensor device which comprises an array of 8×16 pixels, each composed of 4 mini-SiPMs with in situ time-to-digital conversion, a multi-ring network to filter, carry, and process data produced by the sensors at 2Gbps, and a 130nm CMOS process enabling mass-production of photonic modules that are optically interfaced to scintillator crystals. A few tens of sensor devices are tightly abutted on a single PCB to form a so-called sensor tile, thanks to TSV (Through Silicon Via) connections to their backside (replacing conventional wire bonding). The sensor tile is in turn interfaced to an FPGA-based PCB on its back. The resulting photonic module acts as an autonomous sensing and computing unit, individually detecting gamma photons as well as thermal and Compton events. It determines in real time basic information for each scintillation event, such as exact time of arrival, position and energy, and communicates it to its peers in the field of view. Coincidence detection does therefore occur directly in the ring itself, in a differed and distributed manner to ensure scalability. The selected true coincidence events are then collected by a snooper module, from which they are transferred to an external reconstruction computer using Gigabit Ethernet.
We present a digital architecture for fast acquisition of time correlated single photon counting (TCSPC) timestamps from
32×32 CMOS SPAD array. Custom firmware was written to select 64 pixels out of 1024 available for fast transfer of
TCSPC timestamps. Our 64 channel TCSPC is capable of acquiring up to 10 million TCSPC timestamps per second over
a USB2 link. We describe the TCSPC camera (Megaframe), camera interface to the PC and the microscope setup. We
characterize the Megaframe camera for fluorescence lifetime imaging (FLIM) including instrument response function,
time resolution and variability of both across the array. We show a fluorescence lifetime image of a plant specimen
(Convallaria majalis) from a custom-built multifocal multiphoton microscope. The image was acquired in 20 seconds
(with average timestamp acquisition rate of 4.7 million counts per second).
Forster/Fluorescence resonant energy transfer (FRET) has become an extremely important technique to explore
biological interactions in cells and tissues. As the non-radiative transfer of energy from the donor to acceptor occurs
typically only within 1-10nm, FRET measurement allows the user to detect localisation events between protein-conjugated
fluorophores. Compared to other techniques, the use of time correlated single photon counting (TCSPC) to
measure fluorescence lifetime (FLIM) has become the gold standard for measuring FRET interactions in cells. The
technique is fundamentally superior to all existing techniques due to its near ideal counting efficiency, inherent low
excitation light flux (reduced photobleaching and toxicity) and time resolution. Unfortunately due to its slow acquisition
time when compared with other techniques, such as Frequency-domain lifetime determination or anisotropy, this makes
it impractical for measuring dynamic protein interactions in cells. The relatively slow acquisition time of TCSPC FLIM-FRET
is simply due to the system usually employing a single-beam scanning approach where each lifetime (and thus
FRET interaction) is determined individually on a voxel by voxel basis. In this paper we will discuss the development a
microscope system which will parallelize TCSPC for FLIM-FRET in a multi-beam multi-detector format. This will
greatly improve the speed at which the system can operate, whilst maintaining both the high temporal resolution and the
high signal-to-noise for which typical TCPSC systems are known for. We demonstrate this idea using spatial light
modulator (SLM) generated beamlets and single photon avalanche detector (SPAD) array. The performance is evaluated
on a plant specimen.
Fluorescence lifetime imaging microscopy (FLIM) is a well established approach for measuring dynamic signalling
events inside living cells, including detection of protein-protein interactions. The improvement in optical penetration of infrared light compared with linear excitation due to Rayleigh scattering and low absorption have provided imaging
depths of up to 1mm in brain tissue but significant image degradation occurs as samples distort (aberrate) the infrared
excitation beam. Multiphoton time-correlated single photon counting (TCSPC) FLIM is a method for obtaining
functional, high resolution images of biological structures. In order to achieve good statistical accuracy TCSPC typically
requires long acquisition times. We report the development of a multifocal multiphoton microscope (MMM), titled
MegaFLI. Beam parallelization performed via a 3D Gerchberg–Saxton (GS) algorithm using a Spatial Light Modulator
(SLM), increases TCSPC count rate proportional to the number of beamlets produced. A weighted 3D GS algorithm is
employed to improve homogeneity. An added benefit is the implementation of flexible and adaptive optical correction.
Adaptive optics performed by means of Zernike polynomials are used to correct for system induced aberrations. Here we present results with significant improvement in throughput obtained using a novel complementary metal-oxide-semiconductor (CMOS) 1024 pixel single-photon avalanche diode (SPAD) array, opening the way to truly high-throughput FLIM.
A high-speed and hardware-only algorithm using a center of mass method has been proposed for single-detector fluorescence lifetime sensing applications. This algorithm is now implemented on a field programmable gate array to provide fast lifetime estimates from a 32 × 32 low dark count 0.13 μm complementary metal-oxide-semiconductor single-photon avalanche diode (SPAD) plus time-to-digital converter array. A simple look-up table is included to enhance the lifetime resolvability range and photon economics, making it comparable to the commonly used least-square method and maximum-likelihood estimation based software. To demonstrate its performance, a widefield microscope was adapted to accommodate the SPAD array and image different test samples. Fluorescence lifetime imaging microscopy on fluorescent beads in Rhodamine 6G at a frame rate of 50 fps is also shown.
Alignment of a laser to a point source detector for confocal microscopy can be a time-consuming task. The problem is
further exacerbated when multiple laser excitation spots are used in conjunction with a multiple pixel single photon detector;
in addition to X, Y and Z positioning, pixels in a 2D array detector can also be misaligned in roll, pitch and yaw
with respect to each other, causing magnification, rotation and focus variation across the array. We present a technique for
automated multiple point laser alignment to overcome these issues using closed-loop feedback between a laser illuminated
computer controlled Liquid Crystal on Silicon Spatial Light Modulator (LCOS-SLM) acting as the excitation source and a
32×32 pixel CMOS Single Photon Avalanche Diode (SPAD) array as the multiple pixel detection element. The alignment
procedure is discussed and simulated to prove its feasibility before being implemented and tested in a practical optical system.
We show that it is possible to align each independent laser point in a sub-second time scale, significantly simplifying
and speeding up experimental set-up times. The approach provides a solution to the difficulties associated with multiple
point confocal laser alignment to multiple point detector arrays, paving the way for further advances in applications such
as Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Lifetime Imaging Microscopy (FLIM).
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