Tilman Franke,1 Jose L. Serrano-Velez,2,3 Nydia L. Rivera-Rivera,2,3 Eduardo Rosa-Molinarz,3 and atthias Langhorst1 1FEI Munich (Formerly Till Photonics, 2University of Puerto Rico-Rio Piedras, 3University of Puerto Rico-Medical SciencesA fixed-wavelength laser in combination with a nondescanned hyperspectral detection system obtains clean images when used with triple-stained samples with all dyes excited simultaneously. Two-photon microscopy (TPM) is the method of choice for imaging biological samples in vivo, with cellular resolution in three dimensions. Multicolor imaging with several fluorophores – standard in single-photon microscopy – also is desirable in TPM, but existing approaches using multiple lasers are expensive. 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