A wide-field time-domain fluorescence lifetime imaging microscope with optical sectioning

REV SCI INSTRUM 73 (4): 1898-1907 APR 2002 Webb SED, Gu Y, Leveque-Fort S, et al.

This article describes a wide-field time-domain fluorescence lifetime imaging ~FLIM! microscopewith optical sectioning. The FLIM system utilizes a wide-field time-gated optical image intensifier,with a minimum gate width of 85 ps, to achieve high temporal resolution of fluorescence decaysinduced by ultrashort laser pulses. Different configurations, using excitation pulses of picojouleenergy at 80 MHz repetition rate and of nanojoule energy at 10 kHz, are compared. The instrumenthas a temporal dynamic range spanning from 100 ps to tens of ms and is shown to have a temporaldiscrimination better than 10 ps. When applied to laser dye samples, it has produced FLIM mapsdemonstrating sensitivity to variations in both chemical species and local environment, e.g.,viscosity. Wide-field optical sectioning is achieved using the technique of structured illumination,which is applied to remove out-of-focus light that can result in lifetime artifacts. The sectioningstrength, which may be adjusted by choosing an appropriate spatial modulation frequency, ischaracterized and shown to be comparable to that of a confocal microscope. Practical considerationsconcerned with improving the quality of sectioned fluorescence lifetime maps, including using alarge bit depth camera, are discussed.
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