Three dimensional image restoration in fluorescence lifetime imaging microscopy

A microscope set-up and numerical methods are described which enable the me asurement and reconstruction of three-dimensional nanosecond fluorescence l ifetime images in every voxel. The frequency domain fluorescence lifetime i maging microscope (FLIM) utilizes phase detection of high-frequency modulat ed light by homodyne mixing on a microchannel plate image intensifier. The output signal at the image intensifier's phosphor screen is integrated on a charge coupled device camera, A scanning stage is employed to obtain a ser ies of phase-dependent intensity images at equally separated depths in a sp ecimen. The Fourier transform of phase-dependent data gives three-dimension al (3D) images of the Fourier coefficients. These images are deblurred usin g an Iterative Constrained Tikhonov-Miller (ICTM) algorithm in conjunction with a measured point spread function, The 3D reconstruction of fluorescenc e lifetimes are calculated from the deblurred images of the Fourier coeffic ients. An improved spatial and temporal resolution of fluorescence lifetime s was obtained using this approach to the reconstruction of simulated 3D FL IM data. The technique was applied to restore 3D FLIM data of a live cell s pecimen expressing two green fluorescent protein fusion constructs having d istinct fluorescence lifetimes which localized to separate cellular compart ments.