A key part of any super-resolution technique involves accurately correcting for mechanical motion of the sample and setup during acquisition. If left uncorrected, drift degrades the resolution of the final reconstructed image and can introduce unwanted artifacts. Here, we describe how to implement active stabilization, thereby reducing drift to ~1 nm across all three dimensions. In this protocol, we show how to implement our method on custom and standard microscopy hardware. We detail the construction of a separate illumination and detection path, dedicated exclusively to acquiring the diffraction pattern of fiducials deposited on the imaging slide. We also show how to focus lock and adjust the focus in arbitrary nanometer step size increments. Our real-time focus locking is based on kHz calculations performed using the graphics processing unit. The fast calculations allow for rapid repositioning of the sample, which reduces drift below the photon-limited localization precision. Our approach allows for a single-molecule and/or super-resolution image acquisition free from movement artifacts and eliminates the need for complex algorithms or hardware installations. The method is also useful for long acquisitions which span over hours or days, such as multicolor super resolution. Installation does not require specialist knowledge and can be implemented in standard biological laboratories. The full protocol can be implemented within ~2 weeks.

任何超分辨率技术的一个关键部分都涉及在采集过程中准确校正样品的机械运动和设置。如果不进行校正，漂移会降低最终重建图像的分辨率，并可能引入不需要的伪影。在这里，我们描述了如何实现主动稳定，从而在所有三个维度上将漂移减少到 ~1 nm。在此协议中，我们展示了如何在自定义和标准显微镜硬件上实现我们的方法。我们详细介绍了单独的照明和检测路径的构造，专门用于获取沉积在成像幻灯片上的基准的衍射图案。我们还展示了如何以任意纳米步长增量进行焦点锁定和调整焦点。我们的实时焦点锁定基于使用图形处理单元执行的 kHz 计算。快速计算允许快速重新定位样品，从而减少低于光子限制定位精度的漂移。我们的方法允许无运动伪影的单分子和/或超分辨率图像采集，并且无需复杂的算法或硬件安装。该方法还适用于跨越数小时或数天的长时间采集，例如多色超分辨率。安装不需要专业知识，可以在标准生物实验室中实施。完整的协议可在约 2 周内实施。我们的方法允许无运动伪影的单分子和/或超分辨率图像采集，并且无需复杂的算法或硬件安装。该方法还适用于跨越数小时或数天的长时间采集，例如多色超分辨率。安装不需要专业知识，可以在标准生物实验室中实施。完整的协议可在约 2 周内实施。我们的方法允许无运动伪影的单分子和/或超分辨率图像采集，并且无需复杂的算法或硬件安装。该方法还适用于跨越数小时或数天的长时间采集，例如多色超分辨率。安装不需要专业知识，可以在标准生物实验室中实施。完整的协议可在约 2 周内实施。