VIB-KU Leuven Center for Brain & Disease Research, Electron Microscopy Platform & VIB BioImaging Core, O&N5 Herestraat 49 box 602, 3000, Leuven, Belgium.
KU Leuven Department of Neurosciences, Leuven Brain Institute, O&N5 Herestraat 49 box 602, 3000, Leuven, Belgium.
BMC Biol. 2021 Jul 30;19(1):152. doi: 10.1186/s12915-021-01072-7.
Array tomography (AT) is a high-resolution imaging method to resolve fine details at the organelle level and has the advantage that it can provide 3D volumes to show the tissue context. AT can be carried out in a correlative way, combing light and electron microscopy (LM, EM) techniques. However, the correlation between modalities can be a challenge and delineating specific regions of interest in consecutive sections can be time-consuming. Integrated light and electron microscopes (iLEMs) offer the possibility to provide well-correlated images and may pose an ideal solution for correlative AT. Here, we report a workflow to automate navigation between regions of interest.
We use a targeted approach that allows imaging specific tissue features, like organelles, cell processes, and nuclei at different scales to enable fast, directly correlated in situ AT using an integrated light and electron microscope (iLEM-AT). Our workflow is based on the detection of section boundaries on an initial transmitted light acquisition that serves as a reference space to compensate for changes in shape between sections, and we apply a stepwise refinement of localizations as the magnification increases from LM to EM. With minimal user interaction, this enables autonomous and speedy acquisition of regions containing cells and cellular organelles of interest correlated across different magnifications for LM and EM modalities, providing a more efficient way to obtain 3D images. We provide a proof of concept of our approach and the developed software tools using both Golgi neuronal impregnation staining and fluorescently labeled protein condensates in cells.
Our method facilitates tracing and reconstructing cellular structures over multiple sections, is targeted at high resolution ILEMs, and can be integrated into existing devices, both commercial and custom-built systems.
面扫描断层成像（array tomography，AT）是一种高分辨率成像方法，可解析细胞器水平的细微细节，具有提供 3D 体积以显示组织背景的优势。AT 可以以相关方式进行，结合光学显微镜（light microscopy，LM）和电子显微镜（electron microscopy，EM）技术。然而，模态之间的相关性可能是一个挑战，在连续切片中描绘特定的感兴趣区域可能很耗时。集成光和电子显微镜（integrated light and electron microscope，iLEM）提供了提供良好相关图像的可能性，并且可能是相关 AT 的理想解决方案。在这里，我们报告了一种自动导航感兴趣区域的工作流程。
我们使用一种靶向方法，可以对特定的组织特征（如细胞器、细胞过程和细胞核）进行成像，以不同的尺度进行成像，从而能够使用集成光和电子显微镜（iLEM-AT）快速直接地进行相关原位 AT。我们的工作流程基于在初始透射光采集上检测切片边界，该采集作为参考空间来补偿切片之间的形状变化，并且我们应用逐步细化的局部化方法，随着放大倍数从 LM 增加到 EM。通过最小的用户交互，可以自动且快速地获取包含细胞和感兴趣的细胞细胞器的区域，这些区域在不同放大倍数下在 LM 和 EM 模式下相关联，为获得 3D 图像提供了更有效的方法。我们使用 Golgi 神经元染色和细胞内荧光标记蛋白凝聚物提供了我们方法的概念验证和开发的软件工具。
我们的方法促进了在多个切片上对细胞结构进行追踪和重建，针对高分辨率 iLEM，并可以集成到现有的商用和定制系统中。
