Development of a multispectral multiphoton fluorescence lifetime imaging microscopy system using a streak camera

Abstract

We report on the development of a multispectral multiphoton fluorescence lifetime imaging microscopy (MM-FLIM) system that is the combination a streak camera, a prism spectrophotometer, a femtosecond Ti: Sapphire laser and a fluorescence microscope. This system is versatile with multispectral capability, high temporal (10ps) and spatial (0.36μm) resolution and can be used to make 3-dimensional (3D) (x-y-z) multiphoton fluorescence intensity, spectrally resolved intensity and lifetime measurements with a single detector. The system was calibrated with a F-P etalon and a standard fluorescent dye and the lifetime value obtained was in good agreement with the value reported in the literature. Preliminary results suggest that this MM-FLIM system has integrated high temporal, spatial, and spectral resolution fluorescence detection in one microscopy system. Potential applications of this system include multiwell imaging, tissue discrimination, intracellular physiology and fluorescence resonance energy transfer imaging.