Yang Yuhan, Chen Wei, Fan Jiang Lan, Ji Na
Department of Physics, University of California, Berkeley, CA 94720, USA.
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
Biomed Opt Express. 2020 Dec 15;12(1):354-366. doi: 10.1364/BOE.413049. eCollection 2021 Jan 1.
Two-photon fluorescence microscopy has been widely applied to three-dimensional (3D) imaging of complex samples. Remote focusing by controlling the divergence of excitation light is a common approach to scanning the focus axially. However, microscope objectives induce distortion to the wavefront of non-collimated excitation beams, leading to degraded imaging quality away from the natural focal plane. In this paper, using a liquid-crystal spatial light modulator to control the divergence of the excitation beam through a single objective, we systematically characterized the aberrations introduced by divergence control through microscope objectives of NA 0.45, 0.8, and 1.05. We used adaptive optics to correct the divergence-induced-aberrations and maintain diffraction-limited focal quality over up to 800-µm axial range. We further demonstrated aberration-free remote focusing for imaging of neurites and synapses in the mouse brain.
双光子荧光显微镜已广泛应用于复杂样品的三维（3D）成像。通过控制激发光的发散进行远程聚焦是轴向扫描焦点的常用方法。然而，显微镜物镜会使非准直激发光束的波前产生畸变，导致远离自然焦平面时成像质量下降。在本文中，我们使用液晶空间光调制器通过单个物镜控制激发光束的发散，系统地表征了通过数值孔径为0.45、0.8和1.05的显微镜物镜进行发散控制所引入的像差。我们使用自适应光学来校正发散诱导的像差，并在高达800微米的轴向范围内保持衍射极限的聚焦质量。我们进一步展示了用于小鼠大脑中神经突和突触成像的无像差远程聚焦。
