Abstract
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.
Highlights
Fluorescence based imaging techniques have become essential tools for a large variety of disciplines, ranging from cell-biology, medical diagnosis, and pharmacological development to physical sciences
In a typical time-resolved fluorescence lifetime imaging microscopy (FLIM) experiment, the samples with fluorescent markers are illuminated by a pulsed laser and the time-correlated photons emitted from the markers are collected by detectors
Li et al proposed several non-gating time-domain lifetime algorithms and demonstrated video-rate lifetime imaging [14,15] on single-photon avalanche diode (SPAD) plus in-pixel time-correlated single-photon counting cards (TCSPC) arrays [22]
Summary
Fluorescence based imaging techniques have become essential tools for a large variety of disciplines, ranging from cell-biology, medical diagnosis, and pharmacological development to physical sciences. Li et al proposed several non-gating time-domain lifetime algorithms and demonstrated video-rate lifetime imaging [14,15] on single-photon avalanche diode (SPAD) plus in-pixel TCSPC arrays [22]. Recent developments of CMOS SPADs have shown significant improvements in the dead time [26], dark count [27], technology migration to advanced process [28] and pixel miniaturization [29], and quantum efficiency in the longer wavelength region [30] It is expected high resolution CMOS SPAD arrays for ranging applications [31] will soon be applied to FLIM applications. Common FLIM systems usually use iterative linear or non-linear least square methods (LSM), such as Marquardt-Levenberg algorithms, to extract the lifetimes This approach is accurate and suitable for analyzing multi-exponential decays, it is computationally time consuming which makes it unsuitable for real-time applications. Similar to the above-mentioned ‘single-shot’ systems, the imaging speed of the proposed cameras can operate over 100 f/s
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