The Effects of Optical Aberrations to Illumination Beam Thickness in Two-Photon Excitation Microscopes

When performing in vivo imaging of live samples, it is a big challenge to penetrate thick tissues while still maintaining high resolution and a large field of view because of the sample-induced aberrations. These requirements can be met by combining the benefits of two-photon excitation, beam modulation and adaptive optics in an illumination path. However, the relationship between aberrations and the performance of such a microscopy system has never been systematically and comprehensively assessed. Here, two-photon Gaussian and Bessel beams are modulated as illumination beams, and how aberrations affect the thickness of the illumination beams is evaluated. It is found that the thickness variation is highly related to the azimuthal order of Zernike modes. The thickness of the two-photon Gaussian beam is more sensitive to Zernike modes with lower azimuthal order, while the thickness of the two-photon Bessel beam is more sensitive to the higher-azimuthal-order Zernike modes. So, it is necessary to design a new strategy to correct aberrations according to the effects of different Zernike modes in order to maximize the correction capability of correctors and reduce the correction errors for those insensitive Zernike modes. These results may provide important guidance for the design and evaluation of adaptive optical systems in a two-photon excitation microscope.