Correlative light and electron microscopy (CLEM) is a method used to investigate the exact same region in both light and electron microscopy (EM) in order to add ultra-structural information to a light microscopic (usually fluorescent) signal. Workflows combining optical or fluorescent data with electron microscopic images are complex, hence there is a need to communicate detailed protocols and share tips & tricks for successful application of these methods. With the development of volume-EM techniques such as serial blockface scanning electron microscopy (SBF-SEM) and Focussed Ion Beam-SEM, correlation in three dimensions has become more efficient. Volume electron microscopy allows automated acquisition of serial section imaging data that can be reconstructed in three dimensions (3D) to provide a detailed, geometrically accurate view of cellular ultra-structure. In addition, combining volume-EM with high resolution light microscopy (LM) techniques decreases the resolution gap between LM and EM, making retracing of a region of interest and eventual overlays more straightforward. Here, we present a workflow for 3D CLEM on mouse liver, combining high resolution confocal microscopy with SBF-SEM. In this workflow, we have made use of two types of landmarks: (1) near infrared laser branding marks to find back the region imaged in LM in the electron microscope and (2) landmarks present in the tissue but independent of the cell or structure of interest to make overlay images of LM and EM data. Using this approach, we were able to make accurate 3D-CLEM overlays of liver tissue and correlate the fluorescent signal to the ultra-structural detail provided by the electron microscope. This workflow can be adapted for other dense cellular tissues and thus act as a guide for other three-dimensional correlative studies. This article is protected by copyright. All rights reserved.

中文翻译：

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研究肝组织的 3D-CLEM 工作流程

相关光学和电子显微镜 (CLEM) 是一种用于研究光学和电子显微镜 (EM) 中完全相同区域的方法，以便将超微结构信息添加到光学显微镜（通常是荧光）信号中。将光学或荧光数据与电子显微图像相结合的工作流程很复杂，因此需要传达详细的协议并分享成功应用这些方法的提示和技巧。随着体积 EM 技术的发展，例如串行块面扫描电子显微镜 (SBF-SEM) 和聚焦离子束-SEM，三个维度的相关性变得更加有效。体积电子显微镜允许自动采集连续切片成像数据，这些数据可以在三个维度 (3D) 中重建，以提供详细的、细胞超微结构的几何精确视图。此外，将体积 EM 与高分辨率光学显微镜 (LM) 技术相结合，缩小了 LM 和 EM 之间的分辨率差距，使感兴趣区域的回溯和最终覆盖更加直接。在这里，我们提出了在小鼠肝脏上进行 3D CLEM 的工作流程，将高分辨率共聚焦显微镜与 SBF-SEM 相结合。在这个工作流程中，我们使用了两种类型的地标：(1) 近红外激光品牌标记来找回在电子显微镜中 LM 成像的区域和 (2) 存在于组织中但独立于细胞或结构的地标感兴趣的是制作 LM 和 EM 数据的叠加图像。使用这种方法，我们能够对肝脏组织进行准确的 3D-CLEM 叠加，并将荧光信号与电子显微镜提供的超微结构细节相关联。此工作流程可适用于其他致密细胞组织，因此可作为其他三维相关研究的指南。本文受版权保护。版权所有。