Improved nondegenerate multiphoton microscopy and axial registration with a reflective objective

Conventional, degenerate multiphoton microscopy (D-MPM) requires the use of a high-numerical-aperture (NA) objective. Nondegenerate MPM (ND-MPM) imposes the additional demand for precise spatiotemporal overlap of two distinct excitation sources. We demonstrate that the axial focal shift introduced by refractive objective chromatic aberration hinders the spatial requirement of ND-MPM, whereas the use of a reflective objective overcomes this challenge and allows for improved ND excitation efficiency in spite of a lower NA. Moreover, we demonstrate that reflective objective focusing eliminates the axial misregistration of volumetric stacks in traditional D-MPM experiments when multiple excitation wavelengths are used.