Fluorescence imaging and nonlinear coherent optical microscopy can reveal important spatial properties in cells and biological tissues from fixed situations to in vivo dyna- mics. While microscopy can guide interpretation through morphological observations at the sub-micrometric scale, optical imaging cannot directly access the way molecules are organized with given orientations in 3D at the nanoscale. This property, which is important in many processes in biology, from immunology to development biology and mechanobiology, is today most often studied using methods that are not compatible with real time imaging.

We will show that reporting molecular orientational organization down to the nanoscale is made possible using polarization resolved optical microscopy, which takes advanta- ge of the orientation-sensitive coupling between optical excitation fields and molecular transition dipole moments. Remarkably, the non-paraxial fields propagation allowed in high numerical aperture (NA) microscopy permits to access 3D orientation informati- on that is otherwise delicate to access from pure transverse optics. We will describe how high NA optical polarized imaging can provide information on the way molecules are oriented in 3D, including information on their orientational fluctuations. We will describe in particular polarization sensitive approaches in fluorescent single molecule localization microscopy to reveal actin filaments’ organization in dense regions of the cell cytoskeleton, which is generally challenging to image in super resolution imaging. We will describe possible methods to perform polarized microscopy calibration and optical fields-assessment in 3D (i.e. 3D nanoscale polarimetry), and finally discuss the transposition of 3D polarized methodologies to scanning nonlinear optical microscopy for structural imaging in tissues.