A large-field polarisation-resolved laser scanning microscope: applications to CARS imaging

Laser-scanning imaging techniques are frequently used to probe the molecule spatial orientation in a sample of interest by exploiting selection rules depending on the polarisation of the excitation light. For the successful implementation of these techniques the precise control of the polarisation at the sample level is of fundamental importance. Polarisation distortions induced by the optical elements are often the main limitation factor for the maximum size of the field-of-view in polarisation-resolved (PR) laser-scanning microscopy, since for large scanning angles the polarisation distortions may mask the real sample structure. Here we shall demonstrate the implementation of large-field-of-view PR microscopy and show PR CARS imaging of mouse spinal cord thanks to a careful design of the laser-beam optical path. We shall show that this design leads to strongly suppressed distortions and quantify their effects on the final images. Although the focus of this work is on CARS imaging, we stress that the approaches described here can be successfully applied to a wide range of PR laser-scanning techniques. Lay Description The term polarization-resolved laser-scanning microscopy encompasses a broad set of imaging techniques in which the image is created by progressively scanning the field with excitation laser beams and acquiring the optical response of the sample as a function of the incoming-light polarization. These techniques give access to fine structural details, such as the average spatial orientation of selected molecules or bonds, that cannot be resolved with ordinary optical microscopy techniques. Nevertheless, at present they are not particularly widespread as they require a very precise control of the light polarisation at the sample level. Polarisation distortions induced by the optical elements inside the imaging setup are often the main limiting factor for the size of the field of view in polarisation-resolved (PR) laser-scanning microscopy, since for large scanning angles the polarisation distortions may increase dramatically and mask the real sample structure. Here we shall demonstrate the implementation of a large-field-of-view PR coherent-anti-Stokes Raman-scattering (CARS) microscope and apply it to map the distribution and orientation of selected chemical bonds in a mouse spinal cord. Our implementation benefits from a careful design of the laser-beam optical path that leads to strongly suppressed distortions. CARS imaging is a powerful and innovative technique that employs multiple photons to resonantly excite molecular oscillations, producing a coherent signal. This label-free imaging technique is based on chemical contrast, like traditional Raman micro-imaging, but the signal intensity is typically several orders of magnitude stronger than spontaneous Raman, therefore enabling rapid imaging with high signal-to-noise ratio. We stress that while the focus of this work is on CARS imaging, the approaches described here can be successfully applied to a wide range of PR laser-scanning techniques.