Day-Uei Li

Hardware implementation algorithm and error analysis of high-speed fluorescence lifetime sensing systems using center-of-mass method

A new, simple, high-speed, and hardware-only integration-based fluorescence-lifetime-sensing algorithm using a center-of-mass method (CMM) is proposed to implement lifetime calculations, and its signal-to-noise-ratio based on statistics theory is also deduced. Compared to the commonly used iterative least-squares method or the maximum-likelihood-estimation-based, general purpose fluorescence lifetime imaging microscopy (FLIM) analysis software, the proposed hardware lifetime calculation algorithm with CMM offers direct calculation of fluorescence lifetime based on the collected photon counts and timing information provided by in-pixel circuitry and therefore delivers faster analysis for real-time applications, such as clinical diagnosis. A real-time hardware implementation of this CMM FLIM algorithm suitable for a single-photon avalanche diode array in CMOS imaging technology is now proposed for implementation on field-programmable gate array. The performance of the proposed methods has been tested on Fluorescein, Coumarin 6, and 1,8-anilinonaphthalenesulfonate in water/methanol mixture.

On-chip, time-correlated, fluorescence lifetime extraction algorithms and error analysis

A new, simple, and hardware-only fluorescence-lifetime-imaging microscopy (FLIM) is proposed to implement on-chip lifetime extractions, and their signal-to-noise-ratio based on statistics theory is also deduced. The results are compared with Monte Carlo simulations, giving good agreement. Compared with the commonly used iterative least-squares method or the maximum-likelihood-estimation- (MLE-) based, general purpose FLIM analysis software, our algorithm offers direct calculation of fluorescence lifetime based on the collected photon counts stored in on-chip counters and therefore delivers faster analysis for real-time applications, such as clinical diagnosis. Error analysis considering timing jitter based on statistics theory is carried out for the proposed algorithms and is also compared with MLE to obtain optimized channel width or measurement window and bit resolution of the time-to-digital converters for a given accuracy. A multi-exponential, pipelined fluorescence lifetime method based on the proposed algorithms is also introduced. The performance of the proposed methods has been tested on mono-exponential and four-exponential decay experimental data.

A 160×128 single-photon image sensor with on-pixel 55ps 10b time-to-digital converter

Image sensors capable of resolving the time-of-arrival (ToA) of individual photons with high resolution are needed in several applications, such as fluorescence lifetime imaging microscopy (FLIM), Förster resonance energy transfer (FRET), optical rangefinding, and positron emission tomography. In FRET, for example, typical fluorescence lifetime is of the order of 100 to 300ps, thus deep-subnanosecond resolutions are needed in the instrument response function (IRF). This in turn requires new time-resolved image sensors with better time resolution, increased throughput, and lower costs. Solid-state avalanche photodiodes operated in Geiger-mode, or single-photon avalanche diodes (SPADs), have existed for decades [1] but only recently have SPADs been integrated in CMOS. However, as array sizes have grown, the readout bottleneck has also become evident, leading to hybrid designs or more integration and more parallelism on-chip [2,3]. This trend has accelerated with the introduction of SPAD devices in deep-submicron CMOS, that have enabled the design of massively parallel arrays where the entire photon detection and ToA circuitry is integrated on-pixel [4,5].

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.

Hardware implementation and calibration of background noise for an integration-based fluorescence lifetime sensing algorithm

A new integration based fluorescence lifetime imaging microscopy (FLIM) called IEM has been proposed to implement lifetime extraction [J. Opt. Soc. Am. A25, 1190 (2008)]. A real-time hardware implementation of the IEM FLIM algorithm suitable for single photon avalanche diode arrays in nanometer-scale CMOS technology is now proposed. The problems of reduced pixel readout bandwidth and background noise are studied and a calibration method suitable for FPGA implementation is introduced. In particular, the relationship between signal-to-noise ratio and background noise is considered based on statistics theory and compared with a rapid lifetime determination method and maximum-likelihood estimator with-without background correction. The results are also compared with Monte Carlo simulations giving good agreement. The performance of the proposed methods has been tested on monoexponential decay experimental data. The high flexibility, wide range, and hardware friendliness make IEM the best candidate for system-on-chip integration to our knowledge.

Characterization of large-scale non-uniformities in a 20k TDC/SPAD array integrated in a 130nm CMOS process

With the emergence of large arrays of high-functionality pixels, it has become critical to characterize the performance non-uniformity of such arrays. In this paper we characterize a 160×128 array of complex pixels, each with a single-photon avalanche diode (SPAD) and a time-to-digital converter (TDC). A study of the arrays non-uniformities in terms of the timing resolution, jitter, and photon responsivity is conducted for the pixels at various illumination levels, temperatures, and other operating conditions. In the study we found that, in photon-starved operation, the TDCs exhibit a median resolution of 55ps and a standard deviation of 2 ps. The pixels show a median timing jitter of 140ps. Moreover, we measured negligible variations in photon responsivity while changing the number of active pixels. These findings suggest that the image sensor can be used in highly reliable, large-scale, time-correlated measurements of single photons for biological, molecular, and medical applications. The chip is especially valuable for time-resolved imaging, single-photon counting, and correlation-spectroscopy under many realistic operating conditions.

FPGA implementation of a video-rate fluorescence lifetime imaging system with a 32×32 CMOS single-photon avalanche diode array

A new integration based fluorescence lifetime imaging microscopy (FLIM) called IEM has been proposed to implement lifetime calculations [1]. A real-time hardware implementation of this IEM FLIM algorithm suitable for a single photon avalanche diode (SPAD) array in 0.13µm CMOS technology is now implemented on FPGA. A widefield microscope was adapted to accommodate the array and test it on biological applications. Video-rate fluorescence lifetime imaging has been achieved, by performing parallel 32×32 lifetime calculations, realizing the first, compact, and low-cost FLIM camera.