Le Bourdellès Guillaume, Mercier Luc, Roos Johannes, Bancelin Stéphane, Nägerl U Valentin
Univ. Bordeaux, CNRS, IINS, UMR5297, F-33000 Bordeaux, France.
IOGS, CNRS, LP2N, UMR5298, F-33400 Talence, France.
Biomed Opt Express. 2024 Jan 16;15(2):743-752. doi: 10.1364/BOE.510512. eCollection 2024 Feb 1.
The advent of super-resolution microscopy has opened up new avenues to unveil brain structures with unprecedented spatial resolution in the living state. Yet, its application to live animals remains a genuine challenge. Getting optical access to the brain requires the use of a 'cranial window', whose mounting greatly influences image quality. Indeed, the coverslip used for the cranial window should lie as orthogonal as possible to the optical axis of the objective, or else significant optical aberrations occur. In this work, we assess the effect of the tilt angle of the coverslip on STED and two-photon microscopy, in particular, image brightness and spatial resolution. We then propose an approach to measure and reduce the tilt using a simple device added to the microscope, which can ensure orthogonality with a precision of 0.07°.
超分辨率显微镜的出现开辟了新途径，能够在活体状态下以前所未有的空间分辨率揭示脑结构。然而，将其应用于活体动物仍然是一项真正的挑战。要实现对大脑的光学观察，需要使用“颅骨视窗”，其安装方式对图像质量有很大影响。实际上，用于颅骨视窗的盖玻片应尽可能与物镜的光轴正交，否则会出现明显的光学像差。在这项工作中，我们评估了盖玻片倾斜角度对受激发射损耗（STED）显微镜和双光子显微镜的影响，特别是对图像亮度和空间分辨率的影响。然后，我们提出了一种方法，通过在显微镜上添加一个简单装置来测量和减少倾斜，该装置能够确保正交精度达到0.07°。
