Stokes-vector Resolved Multiphoton/Fluorescence Confocal Scanning Microscopy

Abstract

Microscopy techniques based on the measurements of polarimetric contrasts as proved to be an important tool to extract additional information about the organization and orientation of any anisotropic sample. More particularly, it has been proven their efficiency for quantitative methods for materials science and biomedical diagnosis. Among these techniques, we have developed a complete Stokes-vector and Mueller-matrix scanning microscope allowing the acquisition of all the physical effects induced by the interaction of the polarized light with a medium. This interaction can be summarized in a single 4x4 elements Mueller-matrix by comparing the independent coding and decoding polarization states, giving access to physical parameters such as dichroism, birefringence, and scattering, from the macro- to the microscopic scale. In this work, we proposed to image the anisotropic emission of molecules under illumination from multiphoton and fluorescence microscopy available on the same scanning microscope. Using Stokes-Mueller formalism, we demonstrate the potentiality to acquire intensity images combined with the anisotropy factor orientation and the scattering (Degree of Polarization) mapping for both modalities. As proof of principle, we have imaged particular biological structures of interests (collagen fibers and muscles) for thin and thick samples. Combining all this information, we propose to quantify locally the cellular and molecular organization at a different scale, from the microscopic to the nanoscopic level.