Polarization sensitive second harmonic generation (PSHG) imaging can provide useful information which is unreachable
by intensity SHG imaging. Specifically, it can provide geometrical characteristics of the SHG source molecular
architecture. The information is obtained by rotating the excitation linear polarization and by fitting the SHG intensity
variation to a cylindrical symmetry biophysical model. As a result, the ratios of the non-vanishing χ2 tensor elements,
responsible for the SHG conversion, are retrieved. In the end, by assuming a SHG source with dominant
hyperpolarizability, its molecular orientation can be estimated. Here, we developed and used this approach to retrieve
submicron structural information from cultured neurons and to provide estimation on the effective orientation of the
molecular SHG source in axons. For that purpose, the PSHG images of axons were fitted pixel by pixel using an
algorithm based on the above mentioned model. A coefficient of determination of r2>90% was chosen as a filtering
mechanism. For a selected region of interest we then retrieved the pixels' values histogram of the harmonophores'
effective orientations, θe. The distribution was centred at θe=34.93°, with σ=7.62°. These angle values correspond to the
geometrical characteristics of the tubulin heterodimmers forming the microtubules. Modifications on tubulin dimers may
alter θe, σ thus the PSHG optical technique suggests novel quantitative biomarkers able to characterize neurons'
plasticity as well as disease progression.