A workflow for semi-automated volume correlative light microscopy and transmission electron tomography

IF 1.9 4区 工程技术 Q3 MICROSCOPY
Kohki Konishi, Guilherme Neves, Matthew Russell, Masafumi Mimura, Juan Burrone, Roland Fleck
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引用次数: 0

Abstract

Volume Correlative Light and Electron Microscopy (vCLEM) is a powerful method for assessing the ultrastructure of molecularly defined subcellular domains. A central challenge in vCLEM has been the efficient navigation of Regions of Interest (ROIs) across multimodal and multiscale imaging datasets. We developed two key tools to overcome this challenge. First, we developed a multimodal image registration tool (SegReg) that utilizes segmentation of common objects across modalities and uses a Graphical Processing Unit (GPU) for registration of imaging datasets in two and three dimensions. Secondly, we developed a dedicated image viewer to visualize multimodal image registration in three dimensions (NavROI). Here, we demonstrate the integrated use of SegReg and NavROI to navigate large mouse tissue blocks with preserved fluorescent signals to allow selective targeting for TEM tomography of ROIs containing synapses and the cisternal organelle on the proximal region of the axon of a selected pyramidal neuron. By providing real time guidance to precise X-Y trimming of selected ROIs, reliable estimates of cutting depth relative to ROIs and a clear visual navigation of multimodal and multiscale images, our integrated workflow significantly improves the efficiency and accessibility of vCLEM analysis.

Abstract Image

半自动化体相关光学显微镜和透射电子断层扫描的工作流程。
体积相关光电子显微镜(vCLEM)是一种评估分子亚细胞结构域超微结构的有效方法。vCLEM的一个核心挑战是在多模态和多尺度成像数据集上有效导航感兴趣区域(roi)。我们开发了两个关键工具来克服这一挑战。首先,我们开发了一种多模态图像配准工具(SegReg),该工具利用跨模态的通用对象分割,并使用图形处理单元(GPU)在二维和三维空间中对成像数据集进行配准。其次,我们开发了一个专用的图像查看器来可视化三维多模态图像配准(NavROI)。在这里,我们展示了SegReg和NavROI的综合使用,通过保存的荧光信号来导航大的小鼠组织块,从而可以选择性地靶向TEM断层扫描含有突触的roi和位于选定锥体神经元轴突近端区域的池细胞器。通过对选定roi的精确X-Y切边提供实时指导,相对于roi的可靠切割深度估计以及多模态和多尺度图像的清晰视觉导航,我们的集成工作流程显着提高了vCLEM分析的效率和可访问性。
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来源期刊
Journal of microscopy
Journal of microscopy 工程技术-显微镜技术
CiteScore
4.30
自引率
5.00%
发文量
83
审稿时长
1 months
期刊介绍: The Journal of Microscopy is the oldest journal dedicated to the science of microscopy and the only peer-reviewed publication of the Royal Microscopical Society. It publishes papers that report on the very latest developments in microscopy such as advances in microscopy techniques or novel areas of application. The Journal does not seek to publish routine applications of microscopy or specimen preparation even though the submission may otherwise have a high scientific merit. The scope covers research in the physical and biological sciences and covers imaging methods using light, electrons, X-rays and other radiations as well as atomic force and near field techniques. Interdisciplinary research is welcome. Papers pertaining to microscopy are also welcomed on optical theory, spectroscopy, novel specimen preparation and manipulation methods and image recording, processing and analysis including dynamic analysis of living specimens. Publication types include full papers, hot topic fast tracked communications and review articles. Authors considering submitting a review article should contact the editorial office first.
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