Real-time fluorescence lifetime actuation for cell sorting using a CMOS SPAD silicon photomultiplier.

Time-correlated single photon counting (TCSPC) is a fundamental fluorescence lifetime measurement technique offering high signal to noise ratio (SNR). However, its requirement for complex software algorithms for histogram processing restricts throughput in flow cytometers and prevents on-the-fly sorting of cells. We present a single-point digital silicon photomultiplier (SiPM) detector accomplishing real-time fluorescence lifetime-activated actuation targeting cell sorting applications in flow cytometry. The sensor also achieves burst-integrated fluorescence lifetime (BIFL) detection by TCSPC. The SiPM is a single-chip complementary metal-oxide-semiconductor (CMOS) sensor employing a 32×32 single-photon avalanche diode (SPAD) array and eight pairs of time-interleaved time to digital converters (TI-TDCs) with a 50 ps minimum timing resolution. The sensor's pile-up resistant embedded center of mass method (CMM) processor accomplishes low-latency measurement and thresholding of fluorescence lifetime. A digital control signal is generated with a 16.6 µs latency for cell sorter actuation allowing a maximum cell throughput of 60,000 cells per second and an error rate of 0.6%.

A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging.

We demonstrate diffraction limited multiphoton imaging in a massively parallel, fully addressable time-resolved multi-beam multiphoton microscope capable of producing fluorescence lifetime images with sub-50ps temporal resolution. This imaging platform offers a significant improvement in acquisition speed over single-beam laser scanning FLIM by a factor of 64 without compromising in either the temporal or spatial resolutions of the system. We demonstrate FLIM acquisition at 500 ms with live cells expressing green fluorescent protein. The applicability of the technique to imaging protein-protein interactions in live cells is exemplified by observation of time-dependent FRET between the epidermal growth factor receptor (EGFR) and the adapter protein Grb2 following stimulation with the receptor ligand. Furthermore, ligand-dependent association of HER2-HER3 receptor tyrosine kinases was observed on a similar timescale and involved the internalisation and accumulation or receptor heterodimers within endosomes. These data demonstrate the broad applicability of this novel FLIM technique to the spatio-temporal dynamics of protein-protein interaction.

Time-resolved multifocal multiphoton microscope for high speed FRET imaging in vivo.

Imaging the spatiotemporal interaction of proteins in vivo is essential to understanding the complexities of biological systems. The highest accuracy monitoring of protein-protein interactions is achieved using Förster resonance energy transfer (FRET) measured by fluorescence lifetime imaging, with measurements taking minutes to acquire a single frame, limiting their use in dynamic live cell systems. We present a diffraction limited, massively parallel, time-resolved multifocal multiphoton microscope capable of producing fluorescence lifetime images with 55 ps time-resolution, giving improvements in acquisition speed of a factor of 64. We present demonstrations with FRET imaging in a model cell system and demonstrate in vivo FLIM using a GTPase biosensor in the zebrafish embryo.

A study of pile-up in integrated time-correlated single photon counting systems.

Recent demonstration of highly integrated, solid-state, time-correlated single photon counting (TCSPC) systems in CMOS technology is set to provide significant increases in performance over existing bulky, expensive hardware. Arrays of single photon single photon avalanche diode (SPAD) detectors, timing channels, and signal processing can be integrated on a single silicon chip with a degree of parallelism and computational speed that is unattainable by discrete photomultiplier tube and photon counting card solutions. New multi-channel, multi-detector TCSPC sensor architectures with greatly enhanced throughput due to minimal detector transit (dead) time or timing channel dead time are now feasible. In this paper, we study the potential for future integrated, solid-state TCSPC sensors to exceed the photon pile-up limit through analytic formula and simulation. The results are validated using a 10% fill factor SPAD array and an 8-channel, 52 ps resolution time-to-digital conversion architecture with embedded lifetime estimation. It is demonstrated that pile-up insensitive acquisition is attainable at greater than 10 times the pulse repetition rate providing over 60 dB of extended dynamic range to the TCSPC technique. Our results predict future CMOS TCSPC sensors capable of live-cell transient observations in confocal scanning microscopy, improved resolution of near-infrared optical tomography systems, and fluorescence lifetime activated cell sorting.

Time-domain fluorescence lifetime imaging techniques suitable for solid-state imaging sensor arrays.

We have successfully demonstrated video-rate CMOS single-photon avalanche diode (SPAD)-based cameras for fluorescence lifetime imaging microscopy (FLIM) by applying innovative FLIM algorithms. We also review and compare several time-domain techniques and solid-state FLIM systems, and adapt the proposed algorithms for massive CMOS SPAD-based arrays and hardware implementations. The theoretical error equations are derived and their performances are demonstrated on the data obtained from 0.13 µm CMOS SPAD arrays and the multiple-decay data obtained from scanning PMT systems. In vivo two photon fluorescence lifetime imaging data of FITC-albumin labeled vasculature of a P22 rat carcinosarcoma (BD9 rat window chamber) are used to test how different algorithms perform on bi-decay data. The proposed techniques are capable of producing lifetime images with enough contrast.

A high-throughput time-resolved mini-silicon photomultiplier with embedded fluorescence lifetime estimation in 0.13 µm CMOS.

We describe a miniaturized, high-throughput, time-resolved fluorescence lifetime sensor implemented in a 0.13 m CMOS process, combining single photon detection, multiple channel timing and embedded pre-processing of fluorescence lifetime estimations on a single device. Detection is achieved using an array of single photon avalanche diodes (SPADs) arranged in a digital silicon photomultiplier (SiPM) architecture with 400 ps output pulses and a 10% fill-factor. An array of time-to-digital converters (TDCs) with ≈50 ps resolution records up to 8 photon events during each excitation period. Data from the TDC array is then processed using a centre-of-mass method (CMM) pre-calculation to produce fluorescence lifetime estimations in real-time. The sensor is believed to be the first reported implementation of embedded fluorescence lifetime estimation. The system is demonstrated in a practical laboratory environment with measurements of a variety of fluorescent dyes with different single exponential lifetimes, successfully showing the sensor's ability to overcome the classic pile-up limitation of time-correlated single photon counting (TCSPC) by over an order of magnitude.

Video-rate fluorescence lifetime imaging camera with CMOS single-photon avalanche diode arrays and high-speed imaging algorithm.

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.

Fluorescence lifetime biosensing with DNA microarrays and a CMOS-SPAD imager.

Fluorescence lifetime of dye molecules is a sensitive reporter on local microenvironment which is generally independent of fluorophores concentration and can be used as a means of discrimination between molecules with spectrally overlapping emission. It is therefore a potentially powerful multiplexed detection modality in biosensing but requires extremely low light level operation typical of biological analyte concentrations, long data acquisition periods and on-chip processing capability to realize these advantages. We report here fluorescence lifetime data obtained using a CMOS-SPAD imager in conjunction with DNA microarrays and TIRF excitation geometry. This enables acquisition of single photon arrival time histograms for a 320 pixel FLIM map within less than 26 seconds exposure time. From this, we resolve distinct lifetime signatures corresponding to dye-labelled HCV and quantum-dot-labelled HCMV nucleic acid targets at concentrations as low as 10 nM.

Self-aligned charge read-out for InAs nanowire quantum dots.

A highly sensitive charge detector is realized for a quantum dot in an InAs nanowire. We have developed a self-aligned etching process to fabricate in a single step a quantum point contact in a two-dimensional electron gas and a quantum dot in an InAs nanowire. The quantum dot is strongly coupled to the underlying point contact that is used as a charge detector. The addition of one electron to the quantum dot leads to a change of the conductance of the charge detector by typically 20%. The charge sensitivity of the detector is used to measure Coulomb diamonds as well as charging events outside the dot. Charge stability diagrams measured by transport through the quantum dot and charge detection merge perfectly.