Improved nondegenerate multiphoton microscopy and axial registration with a reflective objective

Abstract

Conventional, degenerate multiphoton microscopy (D-MPM) requires the use of a high-numericalaperture (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. Conventional, or degenerate, multiphoton microscopy (D-MPM) relies on the absorption of two or more spatiotemporally overlapped photons of identical energies [1]. Nondegenerate multiphoton microscopy (ND-MPM) combines synchronized pulses from two lasers of different wavelengths (λ 1 and λ 2 ) to excite a fluorophore transition [2,3]. Provided the two pulses arrive to the same location at the same time, the energies of the photons add at an effective excitation wavelength given by λ 3 = 2 λ 1 −1 + λ 2 −1 −1 [4]. ND-MPM has severalunique advantages, including simultaneous multicolor imaging capabilities [4,5], improved signal-to-background ratio [6], and the ability to excite fluorophores with substrateincompatible absorptions at the virtual λ 3 wavelength. Notably, the total excitation of the combined beams is given by the cross-correlation term I 1 + I 2 2 = I 1 2 + I 2 2 + 2I 1 I 2 , where I 1 2 is the excitation profile (i.e., point spread function, or PSF) at λ 1 , I 2 2 dictates the PSF at λ 2 , and 2I 1 I 2 dictates the PSF at λ 3 [4]. The ability to target a fluorophore at λ 3 with ND-MPM, yet probabilistically stimulate fluorescent events at all three excitation pathways, has been shown to achieve a significant increase in excitation efficiency relative to D-MPM [7].Traditional multiphoton objectives are composed of a series of refractive lenses that compensate for one another's aberrations. High-quality objectives are commonly corrected to improve image quality by minimization of spherical aberration, coma, astigmatism, and distortion. Unfortunately, chromatic aberration is unavoidable with refractive objectives, meaning that distinct wavelengths are focused at different optical z-planes. Achromat objectives tailored to minimize this undesired effect can only provide chromatic correction *