High frame rate fluorescence lifetime imaging

A fast time-domain based fluorescence lifetime imaging (FLIM) microscope is presented that can operate at frame rates of hundreds of frames per second. A beam splitter in the detection path of a wide-field fluorescence microscope divides the fluorescence in two parts. One part is optically delayed with respect to the other. Both parts are viewed with a single time-gated intensified CCD camera with a gate width of 5 ns. The fluorescence lifetime image is obtained from the ratio of these two images. The fluorescence lifetime resolution of the FLIM microscope is verified both with dye solutions and fluorescent latex beads. The fluorescence lifetimes obtained from the reference specimens are in good agreement with values obtained from time correlated single photon counting measurements on the same specimens. The acquisition speed of the FLIM system is evaluated with a measurement of the calcium fluxes in neonatal rat myocytes stained with the calcium probe Oregon Green 488-Bapta. Fluorescence lifetime images of the calcium fluxes related to the beating of the myocytes are acquired with frame rates of up to 100 Hz.