Hebisch E, Wagner E, Westphal V, Sieber J J, Lehnart S E
Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Heart Research Center Göttingen, Department of Cardiology & Pulmonology, University Medical Center Göttingen, Göttingen, Germany.
J Microsc. 2017 Aug;267(2):160-175. doi: 10.1111/jmi.12556. Epub 2017 Apr 3.
Multicolour fluorescence imaging by STimulated Emission Depletion (STED) superresolution microscopy with doughnut-shaped STED laser beams based on different wavelengths for each colour channel requires precise image registration. This is especially important when STED imaging is used for co-localisation studies of two or more native proteins in biological specimens to analyse nanometric subcellular spatial arrangements. We developed a robust postprocessing image registration protocol, with the aim to verify and ultimately optimise multicolour STED image quality. Importantly, this protocol will support any subsequent quantitative localisation analysis at nanometric scales. Henceforth, using an approach that registers each colour channel present during STED imaging individually, this protocol reliably corrects for optical aberrations and inadvertent sample drift. To achieve the latter goal, the protocol combines the experimental sample information, from corresponding STED and confocal images using the same optical beam path and setup, with that of an independent calibration sample. As a result, image registration is based on a strategy that maximises the cross-correlation between sequentially acquired images of the experimental sample, which are strategically combined by the protocol. We demonstrate the general applicability of the image registration protocol by co-staining of the ryanodine receptor calcium release channel in primary mouse cardiomyocytes. To validate this new approach, we identify user-friendly criteria, which - if fulfilled - support optimal image registration. In summary, we introduce a new method for image registration and rationally based postprocessing steps through a highly standardised protocol for multicolour STED imaging, which directly supports the reproducibility of protein co-localisation analyses. Although the reference protocol is discussed exemplarily for two-colour STED imaging, it can be readily expanded to three or more colours and STED channels.
基于每个颜色通道不同波长的环形受激发射损耗（STED）激光束的STED超分辨率显微镜进行多色荧光成像需要精确的图像配准。当STED成像用于生物样本中两种或更多种天然蛋白质的共定位研究以分析纳米级亚细胞空间排列时，这一点尤为重要。我们开发了一种强大的后处理图像配准协议，旨在验证并最终优化多色STED图像质量。重要的是，该协议将支持随后在纳米尺度上的任何定量定位分析。从今往后，使用一种分别对STED成像期间存在的每个颜色通道进行配准的方法，该协议能够可靠地校正光学像差和意外的样本漂移。为了实现后一个目标，该协议将来自使用相同光路和设置的相应STED和共聚焦图像的实验样本信息与独立校准样本的信息相结合。结果，图像配准基于一种策略，该策略最大化了实验样本顺序采集图像之间的互相关性，这些图像由该协议进行策略性组合。我们通过对原代小鼠心肌细胞中的兰尼碱受体钙释放通道进行共染色，证明了图像配准协议的普遍适用性。为了验证这种新方法，我们确定了用户友好的标准，若满足这些标准，则支持最佳图像配准。总之，我们通过一种高度标准化的多色STED成像协议，引入了一种用于图像配准和基于合理原则的后处理步骤的新方法，该方法直接支持蛋白质共定位分析的可重复性。尽管参考协议以双色STED成像为例进行了讨论，但它可以很容易地扩展到三种或更多种颜色以及STED通道。
