Rotating-polarization CARS microscopy: combining chemical and molecular orientation sensitivity.

Coherent Anti-Stokes Raman Spectroscopy (CARS) is a non-linear process in which the energy difference of a pair of incoming photons matches the energy of the vibrational mode of a molecular bond of interest. This phonon population is coherently probed by a third photon and anti-Stokes radiation is emitted. Here a novel approach to CARS microscopy is presented yielding the intensity of the anti-Stokes emission, the directionality the molecular bonds of interest, and their average orientation. Myelinated axons in fixed mouse-brain slices have been imaged by RP-CARS. We were able to detect the local average direction of the acylic chains of membrane phospholipids and their spatial anisotropy. This novel method may impact the study of healthy brain circuitry as well as demyelinating diseases or other pathological states associated with altered neural connectivity.