Ultrashort pulse, multispectral nonlinear 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 simultaneously. A 16-channel multispectral detector is used to delineate the multiple nonlinear optical signals, pixel by pixel, in NLOM. The ability to image multiple fluorescent protein mutants and collagen, simultaneously, enables serial measurements of cell-cell and cell-matrix interactions in our 3D tissue model and characterization of fundamental processes in angiogenic morphogenesis.
