May Molly A, Bawart Martin, Langeslag Michiel, Bernet Stefan, Kress Michaela, Ritsch-Marte Monika, Jesacher Alexander
Institute of Biomedical Physics, Medical University of Innsbruck, Müllerstraße 44, 6020 Innsbruck, Austria.
Institute of Physiology, Medical University of Innsbruck, Schöpfstraße 41, 6020 Innsbruck, Austria.
Biomed Opt Express. 2020 Nov 16;11(12):7183-7191. doi: 10.1364/BOE.405863. eCollection 2020 Dec 1.
Fast, volumetric structural and functional imaging of cellular and sub-cellular dynamics inside the living brain is one of the most desired capabilities in the neurosciences, but still faces serious challenges. Specifically, while few solutions for rapid 3D scanning exist, it is generally much easier to facilitate fast in-plane scanning than it is to scan axially at high speeds. Remote focusing in which the imaging plane is shifted along the optical axis by a tunable lens while maintaining the position of the sample and objective is a promising approach to increase the axial scan speed, but existing techniques often introduce severe optical aberrations in high-NA imaging systems, eliminating the possibility of diffraction-limited single-cell imaging. Here, we demonstrate near diffraction-limited, volumetric two-photon fluorescence microscopy in which we resolve the deep sub-micron structures of single microglia cells with axial scanning performed using a novel high-NA remote focusing method. Image contrast is maintained to within 7% compared to mechanical sample stepping and the focal volume remains nearly diffraction-limited over an axial range greater than 86 µm.
对活脑内细胞及亚细胞动力学进行快速、体视结构和功能成像，是神经科学领域最期望具备的能力之一，但仍面临严峻挑战。具体而言，虽然快速三维扫描的解决方案很少，但通常实现快速平面内扫描比高速轴向扫描要容易得多。远程聚焦是一种很有前景的提高轴向扫描速度的方法，即成像平面通过可调透镜沿光轴移动，同时保持样品和物镜的位置不变，但现有技术在高数值孔径成像系统中常常会引入严重的光学像差，从而排除了实现衍射极限单细胞成像的可能性。在此，我们展示了近衍射极限的体视双光子荧光显微镜，其中我们使用一种新型高数值孔径远程聚焦方法进行轴向扫描，分辨出了单个小胶质细胞的深部亚微米结构。与机械样品步进相比，图像对比度保持在7%以内，并且在大于86微米的轴向范围内，焦体积几乎保持衍射极限。
