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

Abstract

A SPAD-based line sensor fabricated in 130 nm CMOS technology capable of acquiring time-resolved fluorescence spectra (TRFS) in 8.3 milliseconds is presented. To the best of our knowledge, this is the fastest time correlated single photon counting (TCSPC) TRFS acquisition reported to date. The line sensor is an upgrade to our prior work and incorporates: i) parallelized interface from sensor to surrounding circuitry enabling high line rate to the PC (19,000 lines/s) and ii) novel time-gating architecture where detected photons in the OFF region are rejected digitally after the output stage of the SPAD. The time-gating architecture was chosen to avoid electrical transients on the SPAD high voltage supplies when gating is achieved by excess bias modulation. The time-gate has an adjustable location and time window width allowing the user to focus on time-events of interest. On-chip integrated center-of-mass (CMM) calculations provide efficient acquisition of photon arrivals and direct lifetime estimation of fluorescence decays. Furthermore, any of the SPC, TCSPC and on-chip CMM modes can be used in conjunction with the time-gating. The higher readout rate and versatile architecture greatly empower the user and will allow widespread applications across many techniques and disciplines. Here we focused on 3 examples of TRFS and time-gated Raman spectroscopy: i) kinetics of chlorophyll A fluorescence from an intact leaf; ii) kinetics of a thrombin biosensor FRET probe from quenched to fluorescence states; iii) ex vivo mouse lung tissue autofluorescence TRFS; iv) time-gated Raman spectroscopy of toluene at 3056 cm peak. To the best of our knowledge, we detect spectrally for the first time the fast rise in fluorescence lifetime of chlorophyll A in a measurement over single fluorescent transient.

Single-photon avalanche diode imagers in biophotonics: review and outlook

“…More recently, Krstajić et al presented a linear 256×2 SPAD array with pixel pitch of 23.7 µm and a high fill factor of 43.7%, implemented in a 130 nm CMOS process [23,65,66]. Each pixel was connected to one TDC with a 40 ps LSB.…”

“…While scanning protein-protein interactions in live cells with a frame time of 500 ms, they studied changes in FRET interactions between epidermal growth factor receptors (EGFR) and adapter proteins Grb2, as well as a ligand-dependent association of HER2-HER3 receptor tyrosine kinases. Kufcsák et al used 5-carboxyfluorescein as a donor and methyl red as an acceptor in FRET for thrombin detection [66]. Thrombin cleaves the connection between the donor and acceptor, separating them in space and removing the energy transfer.…”

“…The linear 256×2 array by Krstajić et al [23], which was already mentioned in the FLIM section, has also been used for surface-enhanced Raman spectroscopy (SERS), again within the PROTEUS project. Significant hardware and software improvements of the same sensor are reported in [66], whereas removal of fluorescent background signals (in addition to DCR) is illustrated in [65].…”

48-spot single-molecule FRET setup with periodic acceptor excitation

“…While larger SPAD arrays have been demonstrated by other groups, they are fabricated using standard high-voltage CMOS processes resulting in poorer photon-counting performance than the custom technology process employed here. Convincing applications for cell FCS and FLIM, among others, have been published with these CMOS SPAD arrays [42][43][44][45][46] , (for a comprehensive review see 47) but they still remain far from providing the sensitivity needed for single-molecule applications.…”

A $64\times64$ SPAD Array for Portable Colorimetric Sensing, Fluorescence and X-Ray Imaging

“…T HE use of CMOS single photon avalanche diodes (SPADs) in biomedical sensing and imaging has increased as a consequence of their performance in terms of fast acquisition, high sensitivity and portability [1].Typical applications include fluorescence lifetime imaging microscopy (FLIM) [2]- [7], fluorescence [8], point-of-care testing (POCT) [9], Raman spectroscopy [10], positron emission tomography (PET) [11], endoscopic TOF PET [6], x-ray sensing [12], time-resolved spectroscopy [13], 3D vision and LIDAR [14]- [20], and quantum imaging [21]. All these applications require high sensitivity and extremely fast timing resolution of the order of picoseconds [22].…”