SignificanceStimulated emission depletion (STED) microscopy has been used to address a wide range of neurobiological questions in optically well-accessible samples, such as cell culture or brain slices. However, the application of STED to deeply embedded structures in the brain of living animals remains technically challenging.AimIn previous work, we established chronic STED imaging in the hippocampus in vivo but the gain in spatial resolution was restricted to the lateral plane. In our study, we report on extending the gain in STED resolution into the optical axis to visualize dendritic spines in the hippocampus in vivo.ApproachOur approach is based on a spatial light modulator to shape the focal STED light intensity in all three dimensions and a conically shaped window that is compatible with an objective that has a long working distance and a high numerical aperture. We corrected distortions of the laser wavefront to optimize the shape of the bottle beam of the STED laser.ResultsWe show how the new window design improves the STED point spread function and the spatial resolution using nanobeads. We then demonstrate the beneficial effects for 3D-STED microscopy of dendritic spines, visualized with an unprecedented level of detail in the hippocampus of a living mouse.ConclusionsWe present a methodology to improve the axial resolution for STED microscopy in the deeply embedded hippocampus in vivo, facilitating longitudinal studies of neuroanatomical plasticity at the nanoscale in a wide range of (patho-)physiological contexts.

中文翻译：

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通过 3D 受激发射耗尽显微镜对活小鼠海马体中的树突棘进行成像

意义受激发射损耗 (STED) 显微镜已被用于解决光学容易获取的样本（例如细胞培养物或脑切片）中的各种神经生物学问题。然而，将 STED 应用于活体动物大脑的深层嵌入结构在技术上仍然具有挑战性。目的在之前的工作中，我们在体内海马体中建立了慢性 STED 成像，但空间分辨率的增益仅限于横向平面。在我们的研究中，我们报告了将 STED 分辨率的增益扩展到光轴以可视化体内海马体中的树突棘。方法我们的方法基于空间光调制器，以在所有三个维度上塑造焦点 STED 光强度，以及与具有长工作距离和高数值孔径的物镜兼容的圆锥形窗口。我们校正了激光波前的畸变，以优化 STED 激光器瓶形光束的形状。结果我们展示了新窗口设计如何使用纳米珠提高 STED 点扩散函数和空间分辨率。然后，我们展示了树突棘 3D-STED 显微术的有益效果，在活小鼠海马体中以前所未有的细节水平可视化。结论我们提出了一种方法来提高 STED 显微术在体内深层嵌入海马体中的轴向分辨率，