EMBL Australia Node in Single Molecule Science, School of Medical Sciences, University of New South Wales, Sydney, Australia.
ARC Centre of Excellence in Advanced Molecular Imaging, University of New South Wales, Sydney, Australia.
Nat Protoc. 2021 Jan;16(1):497-515. doi: 10.1038/s41596-020-00426-9. Epub 2020 Dec 2.
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地校正样品和设置的机械运动。如果不进行校正,漂移会降低最终重建图像的分辨率,并引入不需要的伪影。在这里,我们描述了如何实现主动稳定,从而将所有三个维度的漂移降低到~1nm。在本方案中,我们展示了如何在定制和标准显微镜硬件上实现我们的方法。我们详细介绍了构建单独的照明和检测路径的过程,该路径专门用于获取成像载玻片上沉积的基准的衍射图案。我们还展示了如何进行焦点锁定和以任意纳米步长增量调整焦点。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,从而将漂移降低到光子限制的定位精度以下。我们的方法可用于不受运动伪影影响的单分子和/或超分辨率图像采集,并且无需复杂的算法或硬件安装。该方法对于跨越数小时或数天的长时间采集(如多色超分辨率)也很有用。安装不需要专业知识,并且可以在标准的生物学实验室中实现。完整的方案可以在大约 2 周内实现。