Abstract
The properties of a novel ultra-fast optical imager, Tpx3Cam, were investigated for macroscopic wide-field phosphorescent lifetime imaging (PLIM) applications. The camera is based on a novel optical sensor and Timepix3 readout chip with a time resolution of 1.6 ns, recording of photon arrival time and time over threshold for each pixel, and readout rate of 80 megapixels per second. In this study, we coupled the camera to an image intensifier, a 760 nm emission filter and a 50 mm lens, and with a super-bright 627nm LED providing pulsed excitation of a 18 × 18 mm sample area. The resulting macro-imager with compact and rigid optical alignment of its main components was characterised using planar phosphorescent O2 sensors and a resolution plate mask. Several acquisition and image processing algorithms were evaluated to optimise the system resolution and performance for the wide-field PLIM, followed by imaging a variety of phosphorescent samples. The new PLIM system looks promising, particularly for phosphorescence lifetime-based imaging of O2 in various chemical and biological samples.
Highlights
Fluorescence Lifetime IMaging (FLIM) is gaining popularity in the biological and medical research [1,2,3] since FLIM measurements are less affected by the factors influencing fluorescence intensity such as probe concentration, photobleaching, excitation intensity, optical alignment, scattering and autofluorescence
The classical technique for recording fluorescence lifetimes is Time Correlated Single Photon Counting (TCSPC), in which a pulsed excitation laser is coupled with a single photon counting detector, which uses fast timing electronics to detect the emitted photons and extract the Received 26 Aug 2019; revised 16 Oct 2019; accepted 16 Oct 2019; published 5 Dec 2019
Laser scanning TCSPC-FLIM has advanced in recent years in its optoelectronic components, sensitivity, excitation sources and emission range, and has been implemented in clinical applications, including dermatology [14], ophthalmology [15], and cancer [16]
Summary
Fluorescence Lifetime IMaging (FLIM) is gaining popularity in the biological and medical research [1,2,3] since FLIM measurements are less affected by the factors influencing fluorescence intensity such as probe concentration, photobleaching, excitation intensity, optical alignment, scattering and autofluorescence. The classical technique for recording fluorescence lifetimes is Time Correlated Single Photon Counting (TCSPC), in which a pulsed excitation laser is coupled with a single photon counting detector, which uses fast timing electronics to detect the emitted photons and extract the characteristics of emission decay. Laser scanning TCSPC-FLIM has advanced in recent years in its optoelectronic components, sensitivity, excitation sources and emission range, and has been implemented in clinical applications, including dermatology [14], ophthalmology [15], and cancer [16]. Pixel-by-pixel scanning leads to slow image acquisition times [17,18,19], for PLIM applications where long pixel dwell times are required to record the long-lived phosphorescence
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