A system and method of high-speed microscopy using a two-photon microscope with spectral resolution. The microscope is operable to provide two- to five-dimensional fluorescence images of samples, including two or three spatial dimensions, a spectral dimension (for fluorescence emission), and a temporal dimension (on a scale of less than approximately one second). Two-dimensional (spatial) images with a complete wavelength spectrum are generated from a single scan of a sample. The microscope may include one of a multi-beam point scanning microscope, a single beam line scanning microscope, and a multi-beam line scanning microscope. The line scans may be formed using one or more of curved mirrors and lenses. The multiple beams may be formed using one of a grating, an array of lenses, and a beam splitter.