Whole-section tumor micro-architecture analysis by a two-dimensional phasor-based approach applied to polarization-dependent second harmonic imaging

Second Harmonic Generation (SHG) microscopy has gained much interest in the histopathology field since it allows label-free imaging of tissues simultaneously providing information on their morphology and on the collagen microarchitecture, thereby highlighting the onset of pathologies and diseases. A wide request of image analysis tools is growing, with the aim to increase the reliability of the analysis of the huge amount of acquired data and to assist pathologists in a user-independent way during their diagnosis. In this light, we exploit here a set of phasor-parameters that, coupled to a 2-dimensional phasor-based approach (μMAPPS, Microscopic Multiparametric Analysis by Phasor projection of Polarization-dependent SHG signal) and a clustering algorithm, allow to automatically recover different collagen microarchitectures in the tissues extracellular matrix. The collagen fibrils microscopic parameters (orientation and anisotropy) are analyzed at a mesoscopic level by quantifying their local spatial heterogeneity in histopathology sections (few mm in size) from two cancer xenografts in mice, in order to maximally discriminate different collagen organizations, allowing in this case to identify the tumor area with respect to the surrounding skin tissue. We show that the “fibril entropy” parameter, which describes the tissue order on a selected spatial scale, is the most effective in enlightening the tumor edges, opening the possibility of their automatic segmentation. Our method, therefore, combined with tissue morphology information, has the potential to become a support to standard histopathology in diseases diagnosis