Cells are 3D objects. Therefore, volume EM (vEM) is often crucial for correct interpretation of ultrastructural data. Today, scanning EM (SEM) methods such as focused ion beam (FIB)–SEM are frequently used for vEM analyses. While they allow automated data acquisition, precise targeting of volumes of interest within a large sample remains challenging. Here, we provide a workflow to target FIB-SEM acquisition of fluorescently labeled cells or subcellular structures with micrometer precision. The strategy relies on fluorescence preservation during sample preparation and targeted trimming guided by confocal maps of the fluorescence signal in the resin block. Laser branding is used to create landmarks on the block surface to position the FIB-SEM acquisition. Using this method, we acquired volumes of specific single cells within large tissues such as 3D cultures of mouse mammary gland organoids, tracheal terminal cells in Drosophila melanogaster larvae, and ovarian follicular cells in adult Drosophila, discovering ultrastructural details that could not be appreciated before.

中文翻译：

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体积电子显微镜的高精度靶向工作流程

细胞是 3D 对象。因此，体积 EM (vEM) 通常对于正确解释超微结构数据至关重要。如今，聚焦离子束 (FIB)–SEM 等扫描 EM (SEM) 方法经常用于 vEM 分析。虽然它们允许自动数据采集，但在大样本中精确定位感兴趣的体积仍然具有挑战性。在这里，我们提供了一个工作流程，以微米级精度以 FIB-SEM 采集荧光标记细胞或亚细胞结构。该策略依赖于样品制备过程中的荧光保存以及由树脂块中荧光信号的共焦图引导的有针对性的修剪。激光标记用于在块表面创建地标以定位 FIB-SEM 采集。利用这种方法，我们获得了大型组织中大量的特定单细胞，例如小鼠乳腺类器官的 3D 培养物、果蝇幼虫的气管末端细胞和成年果蝇的卵巢滤泡细胞，发现了以前无法理解的超微结构细节。