Simulation analysis and comparison of the effects of aberrations on multiphoton fluorescent light sheets

Abstract

Multi-photon scanning light-sheet microscope, which combines the advantages of light-sheet illumination and multi-photon excitation, has high axial resolution, low photobleaching, and a large field of view. However, as the light sheet goes deeper into the biological tissue, biological optical aberrations caused by the uneven refractive index inside the organism become non-negligible factors that dramatically increase the thickness and decrease the light intensity of the light sheet. This paper theoretically deduces and simulates the influence of aberrations on the intensity and thickness of the two-, three-, and four-photon fluorescent light sheet. A clear general trend is obvious. With the increasing amplitude of aberration, the effect of aberration on the light sheet thickness and intensity increases in each multiphoton light sheet. In terms of thickness, the impact of high-order aberrations diminishes from the two-photon to the four-photon light sheet. However, in terms of intensity, the impact of high-order aberrations increases from the two-photon to the four-photon light sheet. The number of items to be corrected for each multiphoton light sheet is given and compared under random aberrations with different amplitudes. The results provide important guidance for the design and evaluation of adaptive optics in multi-photon light sheet microscopes.