Abstract
We describe the performance of a new wide area time-gated single-photon avalanche diode (SPAD) array for phasor-FLIM, exploring the effect of gate length, gate number and signal intensity on the measured lifetime accuracy and precision. We conclude that the detector functions essentially as an ideal shot noise limited sensor and is capable of video rate FLIM measurement. The phasor approach used in this work appears ideally suited to handle the large amount of data generated by this type of very large sensor (512 × 512 pixels), even in the case of small number of gates and limited photon budget.
Highlights
Among the many optical imaging modalities available today, fluorescence imaging is popular in biological sciences due to its versatility and specificity
We found that single pixel phasor values corresponding to such noisy pixels are distinguished from the remainder of the pixels as they are randomly scattered in the phasor plot
ATTO 550, Cy3B and Rhodamine 6G (R6G) samples (figures 5(a)–(c)) were aqueous solutions sandwiched between two glass coverslips separated by a 1 mm thick rubber gasket, allowing to test the wide-field response uniformity of the detector
Summary
Among the many optical imaging modalities available today, fluorescence imaging is popular in biological sciences due to its versatility and specificity. Fluorescence imaging can target almost any molecule of interest with minimal interference with the molecule’s function, while a vast range of fluorophores with distinct absorption and emission spectra is available today. For samples characterized by longer lifetimes such as the samples used here, the decays never reach the background level due to the periodic excitation, and a different approach based on a 3-point background estimation needs to be used [29]. While these methods do not mitigate the slight increase in dispersion caused by background photons, they improve the calculated phasor location and subsequent analysis
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