Molecular Biophysics, Department of Physics, Faculty of Science, Utrecht University, Princetonplein 1, 3584 CC Utrecht, Netherlands.
Department of Cell Biology, Centre for Molecular Medicine, University Medical Centre Utrecht, Utrecht, Netherlands.
Ultramicroscopy. 2020 Aug;215:113007. doi: 10.1016/j.ultramic.2020.113007. Epub 2020 May 6.
In correlative light and electron microscopy (CLEM), the capabilities of fluorescence microscopy (FM) and electron microscopy (EM) are united. FM combines a large field of view with high sensitivity for detecting fluorescence, which makes it an excellent tool for identifying regions of interest. EM has a much smaller field of view but offers superb resolution that allows studying cellular ultrastructure. In CLEM, the potentials of both techniques are combined but a limiting factor is the large difference in resolution between the two imaging modalities. Adding super resolution FM to CLEM reduces the resolution gap between FM and EM; it offers the possibility of identifying multiple targets within the diffraction limit and can increase correlation accuracy. CLEM is usually carried out in two separate setups, which requires transfer of the sample. This may result in distortion and damage of the specimen, which can complicate finding back regions of interest. By integrating the two imaging modalities, such problems can be avoided. Here, an integrated super resolution correlative microscopy approach is presented based on a wide-field super resolution FM integrated in a Transmission Electron Microscope (TEM). Switching imaging modalities is accomplished by rotation of the TEM sample holder. First imaging experiments are presented on sections of Lowicryl embedded Human Umbilical Vein Endothelial Cells labeled for Caveolin both with Protein A-Gold, and Alexa Fluor®647. TEM and FM images were overlaid using fiducial markers visible in both imaging modalities with an overlay accuracy of 28 ± 11 nm. This is close to the optical resolution of ~50 nm.
在相关光学显微镜和电子显微镜（CLEM）中，荧光显微镜（FM）和电子显微镜（EM）的功能结合在一起。FM 结合了大视场和高灵敏度的荧光检测，使其成为识别感兴趣区域的绝佳工具。EM 的视场要小得多，但提供了卓越的分辨率，允许研究细胞超微结构。在 CLEM 中，两种技术的潜力都得到了结合，但限制因素是两种成像方式之间分辨率的巨大差异。将超分辨率 FM 添加到 CLEM 中可以缩小 FM 和 EM 之间的分辨率差距；它提供了在衍射极限内识别多个目标的可能性，并可以提高相关性的准确性。CLEM 通常在两个单独的设置中进行，这需要转移样本。这可能导致标本变形和损坏，这可能使找到感兴趣的区域变得复杂。通过集成两种成像方式，可以避免这些问题。在这里，提出了一种基于集成在透射电子显微镜（TEM）中的宽场超分辨率 FM 的集成超分辨率相关显微镜方法。通过旋转 TEM 样品架来切换成像模式。首先在用蛋白 A-金和 Alexa Fluor®647 标记的包埋在 Lowicryl 中的人脐静脉内皮细胞切片上进行了成像实验。使用在两种成像模式中都可见的基准标记来覆盖 TEM 和 FM 图像，重叠精度为 28 ± 11 nm。这接近光学分辨率约 50 nm。
