Reactive oxygen FIB spin milling enables correlative workflow for 3D super-resolution light microscopy and serial FIB/SEM of cultured cells.
Reactive oxygen FIB spin milling enables correlative workflow for 3D super-resolution light microscopy and serial FIB/SEM of cultured cells.
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活性氧FIB旋磨为培养细胞的3D超分辨光学显微镜和连续FIB/SEM提供了相关的工作流程。
DOI：
10.1038/s41598-021-92608-y
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发表时间：
2021-06-23
影响因子：
4.6
通讯作者：
Straw M
中科院分区：
文献类型：
--
作者：
Wang J;Randolph S;Wu Q;Botman A;Schardt J;Bouchet-Marquis C;Nan X;Rue C;Straw M
相关光学和电子显微镜（CLEM）是一个强大的工具，用于定义分子标记的生物标本的超微结构的背景下，特别是当超分辨率荧光显微镜（SRM）用于CLEM。然而，目前的CLEM受到两种模式之间样品制备要求的明显差异的限制。对于使用SRM的CLEM，一种或两种模态的小感兴趣区域（ROI）也导致低成功率和成像吞吐量。为了克服这些局限性，在这里，我们提出了一个CLEM工作流程的基础上，一种新的聚焦离子束/扫描电子显微镜（FIB/SEM）与常见的SRM兼容，用于生物标本成像的3D分辨率和提高成像吞吐量。通过在等离子体FIB（PFIB）中使用活性氧源和旋转样品台，新颖的FIB/SEM能够通过称为氧气连续旋转磨（OSSM）的过程实现对树脂包埋的细胞的数百微米大面积3D分析。与目前的FIB机制相比，OSSM提供了温和的侵蚀，高度一致的切片厚度，减少了SEM成像期间的充电，并提高了SEM对比度，而无需增加后染色和固定的剂量。OSSM-SEM的这些特性使我们能够将其与干涉光活化定位显微镜（iPALM）配对，iPALM是一种最近的SRM技术，可在水合样品上提供10-20 nm的各向同性空间分辨率，用于3D CLEM成像。我们证明了CLEM工作流程可推广到使用其他SRM策略，使用人骨肉瘤（U2 OS）细胞中的线粒体作为模型系统，其中免疫染色的TOM 20，线粒体外膜的标记物，用于iPALM。由于OSSM-SEM的大扫描面积，现在可以根据iPALM的需要选择尽可能多的FOV，并在EM中方便地重新定位它们，这提高了成像吞吐量。后固定剂量的显著降低也有助于更好地保存样品超微结构，如通过OSSM-SEM和iPALM图像之间的出色3D配准以及通过TOM 20（通过iPALM）到线粒体外围的准确定位（通过OSSM-SEM）所证明的。这些优点使OSSM-SEM成为CLEM应用的理想模式。由于OSSM-SEM仍在开发中，我们还讨论了一些剩余的问题和与SEM单独或与CLEM的生物成像的影响。
Correlative light and electron microscopy (CLEM) is a powerful tool for defining the ultrastructural context of molecularly-labeled biological specimens, particularly when superresolution fluorescence microscopy (SRM) is used for CLEM. Current CLEM, however, is limited by the stark differences in sample preparation requirements between the two modalities. For CLEM using SRM, the small region of interest (ROI) of either or both modalities also leads to low success rate and imaging throughput. To overcome these limitations, here we present a CLEM workflow based on a novel focused ion beam/scanning electron microscope (FIB/SEM) compatible with common SRM for imaging biological specimen with ultrahigh 3D resolution and improved imaging throughput. By using a reactive oxygen source in a plasma FIB (PFIB) and a rotating sample stage, the novel FIB/SEM was able to achieve several hundreds of micrometer large area 3D analysis of resin embedded cells through a process named oxygen serial spin mill (OSSM). Compared with current FIB mechanisms, OSSM offers gentle erosion, highly consistent slice thickness, reduced charging during SEM imaging, and improved SEM contrast without increasing the dose of post-staining and fixation. These characteristics of OSSM-SEM allowed us to pair it with interferometric photoactivated localization microscopy (iPALM), a recent SRM technique that affords 10–20 nm isotropic spatial resolution on hydrated samples, for 3D CLEM imaging. We demonstrate a CLEM workflow generalizable to using other SRM strategies using mitochondria in human osteosarcoma (U2OS) cells as a model system, where immunostained TOM20, a marker for the mitochondrial outer membrane, was used for iPALM. Owing to the large scan area of OSSM-SEM, it is now possible to select as many FOVs as needed for iPALM and conveniently re-locate them in EM, this improving the imaging throughput. The significantly reduced dose of post-fixation also helped to better preserve the sample ultrastructures as evidenced by the excellent 3D registration between OSSM-SEM and iPALM images and by the accurate localization of TOM20 (by iPALM) to the peripheries of mitochondria (by OSSM-SEM). These advantages make OSSM-SEM an ideal modality for CLEM applications. As OSSM-SEM is still in development, we also discuss some of the remaining issues and the implications to biological imaging with SEM alone or with CLEM.
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影响因子：
2
作者：
Kremer A;Lippens S;Bartunkova S;Asselbergh B;Blanpain C;Fendrych M;Goossens A;Holt M;Janssens S;Krols M;Larsimont JC;Mc Guire C;Nowack MK;Saelens X;Schertel A;Schepens B;Slezak M;Timmerman V;Theunis C;VAN Brempt R;Visser Y;Guérin CJ
通讯作者：
Guérin CJ
影响因子：
4.6
作者：
Johnson E;Seiradake E;Jones EY;Davis I;Grünewald K;Kaufmann R
通讯作者：
Kaufmann R
DOI：
10.1073/pnas.1121558109
发表时间：
2012-04-17
影响因子：
11.1
作者：
Kopek, Benjamin G.;Shtengel, Gleb;Hess, Harald F.
通讯作者：
Hess, Harald F.
影响因子：
4.6
作者：
Martin AA;Randolph S;Botman A;Toth M;Aharonovich I
通讯作者：
Aharonovich I
影响因子：
64.8
作者：
Dickson, RM;Cubitt, AB;Moerner, WE
通讯作者：
Moerner, WE