Abstract
Wide-field time-correlated single photon counting detection techniques, where the position and the arrival time of the photons are recorded simultaneously using a camera, have made some advances recently. The technology and instrumentation used for this approach is employed in areas such as nuclear science, mass spectroscopy and positron emission tomography, but here, we discuss some of the wide-field TCSPC methods, for applications in fluorescence microscopy. We describe work by us and others as presented in the Ulitima fast imaging and tracking conference at the Argonne National Laboratory in September 2018, from phosphorescence lifetime imaging (PLIM) microscopy on the microsecond time scale to fluorescence lifetime imaging (FLIM) on the nanosecond time scale, and highlight some applications of these techniques.
Highlights
The fluorescence lifetime is the average time a fluorophore remains in the excited state, typically nanoseconds
Fluorescence lifetime imaging (FLIM) and phosphorescence lifetime imaging (PLIM) can measure the lifetimes in every pixel, providing image contrast according to the lifetime, which provides contrast according to viscosity, oxygen or ion concentration or temperature, depending on the type of fluorescent probe employed
While time-correlated single photon counting (TCSPC) is straight-forward to implement with scanning microscopy, there are a number of fluorescence microscopy methods that employ a camera, for example lightsheet microscopy, total internal reflection fluorescence (TIRF), supercritical angle fluorescence and super-resolution fluorescence microscopy methods based on localisation of fluorophores
Summary
The fluorescence lifetime is the average time a fluorophore remains in the excited state, typically nanoseconds. Wide-field time-correlated single photon counting detection techniques, where the position and the arrival time of the photons are recorded simultaneously using a camera, have made some advances recently. The technology and instrumentation used for this approach is employed in areas such as nuclear science, mass spectroscopy and positron emission tomography, but here, we discuss some of the wide-field TCSPC methods, for applications in fluorescence microscopy.
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