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

Abstract

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%.

Towards sensitive, high-throughput, biomolecular assays based on fluorescence lifetime

“…To the best of our knowledge, no available commercial TDC allows such real-time data processing which requires hardware programming to achieve the desired processing rates. (38,58) In conclusion, the technical implications of the present work are twofold. First it demonstrates that FLT detection is achievable with good precision in very small volume samples and chromophore quantities at high detection throughput.…”

“…DWR, however, several applications of TCSPC were recently demonstrated for accurate FLT detection under high throughput conditions. (36)(37)(38) In particular, using a microfluidic flow cytometer designed for efficient FLT detection by TCSPC at a throughput up to 3000 particles per minute, Nedbal et al (37) reported a comprehensive series of experiments demonstrating the superiority of FLT over FLINT for biomolecular interaction sensing in individual cells, based on the detection of FRET (Förster Resonant Energy Transfer) between ns-lived chromophores including fluorescent proteins. We instead have implemented TCSPC in microfluidic droplets and investigated the fundamental limitations imposed by the single photon detection technique on the photon acquisition rate and on the achievable signal-to-noise ratio in high throughput conditions.…”

Time-resolved spectroscopy at 19,000 lines per second using a CMOS SPAD line array enables advanced biophotonics applications

“…This allows photon efficient processing of photon arrival times, because all processing is done on the sensor itself. This technique provides fast fluorescence decay analysis [40][41][42][43], while each operating mode of the sensor can be amended with an adjustable ON (allowing recording of photon events only during the time-gate) or OFF time-gate (register all photon events but the ones during the time-gate).…”

Plug Silicon Photomultiplier for the & Imaging PET System: Physics, Technological Challenges and Application to Modern Nuclear Medicine

“…The implementation of SiPM and SPAD sensitive cells in standard CMOS technology allows the combined integration of sensor and read-out electronics on the same device, with a significant reduction of power consumption and simplification of the operational conditions. In recent years a large number of advanced digital imagers and innovative concepts for light detection were proposed on this basis [15][16][17][18].…”