In biomedical research intravital two-photon fluorescence microscopy has provided insightful information on dynamic processes in vivo. However the use of exogenous labeling agents limits its applications. I will first demonstrate in vivo mouse mast cells imaging using endogenous tryptophan as the fluorophore for immunological research. Laser beam scanning is required in most current two-photon microscopes to achieve 2D images. Based on temporal focusing of femtosecond laser pulses a new type of two-photon microscope can achieve 2D images without scanning the laser beam; therefore it could reach hundreds frames/second imaging speed. To acquire depth information, most modalities still need to move the sample stage mechanically. In temporal focusing two-photon microscope changing the group velocity dispersion of the femtosecond laser pulses could lead to the displacement of the plane of the temporal focus along the optical axis from the geometrical focus of the objective lens, yielding z-scanning as a function of dispersion. Currently my group is developing pulse shaping technique with spatial light modulators (e.g. acousto-opto modulators) to electronically control the dispersion of the femtosecond laser pulses in spectral domain to achieve fast 3D fluorescence imaging.