We applied Two-photon Excited Fluorescence (TPEF), Second/Third Harmonic Generation (SHG and THG) and Fluorescence Lifetime Imaging (FLIM) Non Linear Optics (NLO) Laser-Scanning Microscopy within the same imaging platform to evaluate their use as a diagnostic tool in ovarian tumors. We assess of applicability of this multimodal approach to perform a pathological evaluation of serous and mucinous tumors in human samples. The combination of TPEF-SHG-THG imaging provided complementary information about the interface epithelium/stromal, such as the transformation of epithelium surface (THG) and the overall fibrillar tissue architecture (SHG). The fact that H&E staining is the standard method used in clinical pathology and that the stored samples are usually fixed makes it important a re-evaluation of these samples with NLO microscopy to compare new results with a library of already existing samples. FLIM, however, depends on the chemical environment around the fluorophors that was completely changed after fixation; therefore it only makes sense in unstained samples. Our FLIM results in unstained samples demonstrate that it is possible to discriminate healthy epithelia from serous or mucinous epithelia. Qualitative and quantitative analysis of the different imaging modalities used showed that multimodal nonlinear microscopy has the potential to differentiate between cancerous and healthy ovarian tissue. © 2012 Copyright Society of Photo-Optical Instrumentation Engineers (SPIE).8226 The Society of Photo-Optical Instrumentation Engineers (SPIE),Becker and Hickel GmbH,Boston Electronics Corp.,Carl Zeiss MicroImaging GmbH,Chroma Technologies Corp.Gubbels, J.A.A., Claussen, N., Kapur, A.K., Connor, J.P., Patankar, M.S., The detection, treatment, and biology of epithelial ovarian cancer (2010) J. Ovarian. 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