Second-harmonic generation holography with polarization multiplexing for label-free collagen characterization and imaging
Digital holography is an interference-based imaging technique capable of recording both the amplitude and phase of an electromagnetic field. It can be obtained at the laser illumination wavelength, but also with second-harmonic generation, since the latter is produced in a coherent process. Here we describe the development of a harmonic holographic microscope for 3D single-shot mapping of second-harmonic emitters. The knowledge of the scattered field, in amplitude and phase, in a given plane, that of the camera, allows its reconstruction in any other plane using the angular spectrum representation of the optical fields, a process called 3D numerical back-propagation. In order to probe the polarization dependence of the sample nonlinear response, we implement polarization multiplexing, in which a Wollaston prism creates two off-axis reference beams with orthogonal polarizations and non-parallel propagation directions. Each reference only interferes with the corresponding polarization component in the sample SHG emission, thus providing two independent sets of interference fringes which are easily separated in the angular spectrum representation. From a single measurement, two second-harmonic fields corresponding to orthogonal polarizations can be back-propagated. In the particular case of collagen, the second-harmonic polarization state can reveal the orientation or disorder of molecules and fibers. We demonstrate the feasibility of the method by reconstructing the spatial distribution of the second-harmonic field generated by collagen fibers in a rat-tail tendon sample and show that polarization-multiplexed holography can provide single-shot 3D mapping of biophysical parameters such as the helical pitch angle of collagen molecules.