BACKGROUND Label-free optical techniques such as optical coherence tomography and Raman techniques are reported to hold promise for improved surgical management of central nervous system (CNS) tumors because of their high resolution and intra-operative applicability in fresh tissue samples. We have recently reported on third harmonic generation (THG) imaging as an alternative, label-free tool for fast imaging of histologic characteristics in fresh, unprocessed human tissues. AIM: To determine the potential of THG microscopy for assessment of tumor load in unprocessed tissue samples of patients with a diffuse glioma. MATERIALS & METHODS Freshly obtained CNS tissue samples (n = 55) of 23 patients with diffuse glioma were analyzed using THG microscopy and subsequently processed for routine pathological analysis. Later on, the histology as seen in H&E-stained sections of these samples was correlated with the THG images. Furthermore, using automated image analysis, an algorithm was developed for classification of the THG images as tumor versus non-tumor in order to further facilitate exploitation of THG microscopy as a real-time, intra-operative diagnostic support tool. RESULTS In all samples, THG microscopy revealed histology-grade characteristics with information on a spectrum of cellular and extracellular components. In most tumor-containing samples, THG imaging readily allowed for visualization of (a gradient of invasive) tumor cells in the CNS parenchyma. Interpretation of the THG images by automated, quantitative image analysis, with particular emphasis on cell density and composition of the neuropil, provided an observer-independent interpretation of the same images. While in depth comparative analysis is still ongoing, preliminary results of comparison of (qualitative and quantitative) information in THG and H&E images are very promising. CONCLUSIONS: THG microscopy enables intra-operative visualization of histopathological characteristics in unprocessed human brain tissue. Qualitative and quantitative analysis of THG images of ex-vivo (and ultimately in-situ) brain tissue has great potential for improving CNS tumor surgery.