Fluorescence lifetime imaging microscopy: Two-dimensional distribution measurement of fluorescence lifetime

A newly developed fluorescence lifetime imaging microscopy (FLIM) system has combined the high-temporal resolution of a streak camera with the high-spatial resolution of a microscope to obtain a two-dimensional distribution of fluorescence lifetimes within living cells. The temporal resolution is as short as 20 ps. The effective field of view is 48 x 45 mu m with 0.2 mu m resolution using a 60x water immersion objective. Image acquisition time is as short as 3 s per image. Measured and published values of lifetime for standard fluophores are shown with good agreement. Examples of FLIM and fluorescence resonance energy transfer images are presented.