Ultrashort pulse, multispectral non-linear optical microscopy (NLOM) is developed and used to image, simultaneously, a mixed population of cells expressing different fluorescent protein mutants in a 3D tissue model of angiogenesis. Broadband, sub-10-fs pulses are used to excite multiple fluorescent proteins and generate second harmonic in collagen. A 16-channel multispectral detector is used to delineate the multiple non-linear optical signals, pixel by pixel, in NLOM. The ability to image multiple fluorescent protein mutants and collagen, enables serial measurements of cell-cell and cell-matrix interactions in our 3D tissue model and characterization of fundamental processes in angiogenic morphogenesis.