Department of Biotechnology and Biophysics, University of Würzburg, Biocenter, Am Hubland, Würzburg, Germany.
Department of Biotechnology and Biophysics, University of Würzburg, Biocenter, Am Hubland, Würzburg, Germany; Department of Neurology & Neurosurgery, Montréal Neurological Institute, Montréal, Québec, Canada; Department of Chemistry, McGill University, Montréal, Québec, Canada.
Biophys J. 2019 Jun 4;116(11):2073-2078. doi: 10.1016/j.bpj.2019.04.029. Epub 2019 May 3.
We introduce a method for registration and visualization of correlative super-resolution microscopy images from different microscopy techniques. We established an automated registration procedure based on the generalized Hough transform. We developed a software tool to apply this algorithm and visualize correlated images from structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). To demonstrate the potential of this super-resolution correlator, we visualize the distribution of the presynaptic protein bassoon in the active zones of synapses in the molecular layer of the mouse cerebellum. First, a multiple labeled sample is imaged by SIM, followed by imaging of one of the fluorescent labels by dSTORM. To avoid the use of artificial fiducial markers, we used the signal of Alexa Fluor 647 recorded in switching buffer on the two microscopes for image superposition. We recorded multicolor SIM images in 20-μm thick brain slices to identify synapses in the dendritic system of Purkinje cells and put higher-resolved dSTORM images of the synaptic distribution of bassoon in registry.
我们介绍了一种用于注册和可视化来自不同显微镜技术的相关超分辨率显微镜图像的方法。我们基于广义霍夫变换建立了一种自动注册程序。我们开发了一个软件工具来应用该算法，并可视化结构光照明显微镜 (SIM) 和直接随机光学重建显微镜 (dSTORM) 的相关图像。为了展示这种超分辨率相关器的潜力，我们可视化了在小鼠小脑分子层的突触的活性区中的突触前蛋白 bassoon 的分布。首先，通过 SIM 对多个标记的样本进行成像，然后通过 dSTORM 对其中一个荧光标记进行成像。为了避免使用人工基准标记，我们使用在两台显微镜上的切换缓冲液中记录的 Alexa Fluor 647 信号进行图像叠加。我们在 20-μm 厚的脑切片中记录多色 SIM 图像，以识别浦肯野细胞树突系统中的突触，并将 bassoon 的突触分布的高分辨率 dSTORM 图像注册到其中。
