Sample preparation strategies for efficient correlation of 3D SIM and soft X-ray tomography data at cryogenic temperatures
Sample preparation strategies for efficient correlation of 3D SIM and soft X-ray tomography data at cryogenic temperatures
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DOI：
10.1038/s41596-021-00522-4
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发表时间：
2021-05-14
期刊：
影响因子：
14.8
通讯作者：
Harkiolaki, Maria
中科院分区：
文献类型：
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作者：
Okolo, Chidinma A.;Kounatidis, Ilias;Harkiolaki, Maria
该协议描述了相关的3D冷冻结构照明显微镜和冷冻软X射线断层扫描的样品制备策略。作者还提供了一个直接的比较和建议，关于选择和使用的基准3D相关。3D相关显微镜方法已经彻底改变了生物医学研究，允许获取多维信息，以获得深入了解生物系统。随着相关低温保存方法的出现，低温保存的样品的相关成像已经导致在苛刻的成像机制（例如电子和软X射线断层扫描）下的纳米分辨率成像（2-50 nm）。这些方法现在已与低温下的常规和超分辨率荧光成像相结合，以增加给定样本的信息内容，从而立即需要促进显微镜之间轻松、明确的互相关的协议。我们在这里提出的样品制备策略和不同的工作基准制度，促进三维相关的冷冻结构照明显微镜和冷冻软X射线断层扫描的直接比较。我们的协议已在两个同步加速器光束线（B24在钻石光源在英国和BL 09米斯特拉尔在ALBA在西班牙）进行了测试，并导致了一个决策援助，促进实验设计的战略使用标记的项目要求的基础上的发展。该方案需要1.5小时至3.5天才能完成，具体取决于所用的细胞群（贴壁细胞可能需要数天才能在样品载体上生长）。
This protocol describes sample-preparation strategies for correlative 3D cryo-structured illumination microscopy and cryo-soft X-ray tomography. The authors also provide a direct comparison and recommendations regarding the selection and use of fiducials for 3D correlation.3D correlative microscopy methods have revolutionized biomedical research, allowing the acquisition of multidimensional information to gain an in-depth understanding of biological systems. With the advent of relevant cryo-preservation methods, correlative imaging of cryogenically preserved samples has led to nanometer resolution imaging (2-50 nm) under harsh imaging regimes such as electron and soft X-ray tomography. These methods have now been combined with conventional and super-resolution fluorescence imaging at cryogenic temperatures to augment information content from a given sample, resulting in the immediate requirement for protocols that facilitate hassle-free, unambiguous cross-correlation between microscopes. We present here sample preparation strategies and a direct comparison of different working fiducialization regimes that facilitate 3D correlation of cryo-structured illumination microscopy and cryo-soft X-ray tomography. Our protocol has been tested at two synchrotron beamlines (B24 at Diamond Light Source in the UK and BL09 Mistral at ALBA in Spain) and has led to the development of a decision aid that facilitates experimental design with the strategic use of markers based on project requirements. This protocol takes between 1.5 h and 3.5 d to complete, depending on the cell populations used (adherent cells may require several days to grow on sample carriers).