Fluorescence lifetime three-dimensional microscopy with picosecond precision using a multifocal multiphoton microscope

The combination of pulsed-mode excitation multifocal multiphoton microscopy with a high-repetition, time-gated intensified CCD camera enables efficient three-dimensional (3D) fluorescence lifetime imaging. With a 200-ps gate opening at 76 MHz repetition rate, fluorescence decay can be traced in a sequence of images with varying delays between pulse and gate. Fluorophore lifetimes are measured with a precision of a few picoseconds. As an application we show that, upon two-photon excitation at 800 nm, certain pollen samples feature a multiexponential fluorescence relaxation. Our results indicate that efficient four-dimensional microscopy with hundreds of nanometer spatial and tens of picoseconds temporal resolution is within reach.