μMAPPS: a novel phasor approach to second harmonic analysis for in vitro-in vivo investigation of collagen microstructure

Second Harmonic Generation (SHG) is a label-free imaging method used to monitor collagen organization in tissues. Due to its sensitivity to the incident polarization, it provides microstructural information otherwise unreachable by other intensity based imaging methods. We develop and test a Microscopic Multiparametric Analysis by Phasor projection of Polarization-dependent SHG (mu MAPPS) that maps the features of the collagen architecture in tissues at the micrometer scale. mu MAPPS retrieves pixel-by-pixel the collagen fibrils anisotropy and orientation by operating directly on two coupled phasor spaces, avoiding direct fitting of the polarization dependent SHG signal. We apply mu MAPPS to fixed tissue sections and to the study of the collagen microscopic organization in tumors ex-vivo and in-vivo. We develop a clustering algorithm to automatically group pixels with similar microstructural features. mu MAPPS can perform fast analyses of tissues and opens to future applications for in-situ diagnosis of pathologies and diseases that could assist histo-pathological evaluation.