Super-resolution multiphoton frequency-domain fluorescence lifetime imaging microscopy by generalized stepwise optical saturation (GSOS)

We present the first experimental demonstration of super-resolution multiphoton frequency-domain (FD) fluorescence lifetime imaging microscopy (FLIM). This is obtained through a novel microscopy technique called generalized stepwise optical saturation (GSOS). GSOS utilizes the linear combination of M steps of raw images to improve the imaging resolution by a factor of root M. Here, a super-resolution multiphoton FD-FLIM is demonstrated on various samples, including fixed cells and biological tissues, with a custom-built two-photon FD-FLIM microscope. We demonstrate simultaneous super-resolution intensity and fluorescence lifetime images of a variety of cell cultures and ex vivo tissues. Combined with multiphoton excitation, the proposed GSOS microscopy is able to generate super-resolution FLIM images deep in scattering samples.