Two-photon laser scanning microscopy has become a standard to map thick and live tissues. However, its application for fast and multicolor imaging remains challenging. To address this issue, we report on the implementation of mixed wavelength excitation in a two-photon light-sheet microscope. We illustrate the potential of the technique by recording sustained multicolor two-photon movies of the beating heart in zebrafish embryos with negligible photobleaching at 28 million pixels/second. In particular, 3D reconstructions of the heart periodic motion are obtained with sufficient spatiotemporal resolution to track the fast movements of individual cells during a cardiac cycle. © (2015) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.