Early detection and typing of tumors is pressing matter in clinical research with important impacts for prognosis and
successful treatment. Currently, staining is the golden standard in histopathology but requires surgical removal of tissue.
In order to avoid resection of non-diseased tissue a non-invasive real-time imaging method is required which can be
applied ideally intrasurgically. In this proceeding a combination of second harmonic generation (SHG), two photon
excited fluorescence (TPEF) and coherent anti-Stokes Raman (CARS) imaging has been employed to investigate tissue
sections of head and neck carcinomas focussing on laryngeal carcinoma. Primary laryngeal and other head and neck
carcinomas consist to 99% of squamous cell carcinoma. By fusing the various imaging methods it is possible to measure
the thickness of the epithelial cell layer as a marker for dysplastic or cancerous tissue degradation and to differentiate
keratinizing and nonkeratininzing squamous cell carcinomas (SCC). As nonkeratinizing SCCs of the oropharynx
correlate with a human papillomavirus (HPV) infection as a subentity of head and neck cancer, and HPV related tumors
are associated with a better clinical prognosis, the differentiation between keratinizing and non-keratinizing forms of
SCCs is of high diagnostic value. TPEF is capable of displaying cell nuclei, therefore, morphologic information as cell
density, cell to cytoplasm ratio, size and shape of cell nuclei can be obtained. SHG - on the other hand - selectively
reveals the collagen matrix of the connective tissue, which is useful for determination of tumor-islets boundaries within
epithelial tissue - a prerequisite for precise resection. Finally CARS in the CH-stretching region visualizes the lipid
content of the tissue, which can be correlated with the dysplastic grade of the tissue.
Multimodal nonlinear imaging constitutes a contemporary approach to investigate the morphochemistry of complex
samples noninvasively and without administration of external labels. Here we discuss our recent success in jointly using
various nonlinear microspectroscopic approaches such as coherent anti-Stokes Raman scattering (CARS), two-photon
fluorescence (TPF) and second-harmonic generation (SHG) to study the chemical composition of surgically removed
tissue sections from laryngeal carcinoma. In particular it will be shown how multimodal nonlinear imaging can be
employed to study the structural and chemical development of disease formation as well as to monitor the clinically
important aspect of tumor boundary detection.