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.

中文翻译：

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高NA两光子单细胞成像，使用衍射可调透镜实现远程聚焦

快速，体积结构和功能成像的活脑内部细胞和亚细胞动力学是神经科学中最需要的功能之一，但仍然面临严峻挑战。具体地说，尽管很少有用于快速3D扫描的解决方案，但是与快速轴向扫描相比，促进平面内快速扫描通常要容易得多。远程聚焦是通过可调透镜使成像平面沿光轴移动，同时保持样品和物镜的位置，是提高轴向扫描速度的一种有前途的方法，但是现有技术通常会在高NA成像中引入严重的光学像差。系统，消除了衍射限制的单细胞成像的可能性。在这里，我们展示了近衍射极限，体积两光子荧光显微镜，其中我们通过使用新型高NA远程聚焦方法进行的轴向扫描来解析单个小胶质细胞的深亚微米结构。与机械样品步进相比，图像对比度保持在7％之内，并且在大于86 µm的轴向范围内，焦距几乎保持衍射极限。