Elferich Johannes, Kaminek Marek, Kong Lingli, Odriozola Adolfo, Kukulski Wanda, Zuber Benoît, Grigorieff Nikolaus
Howard Hughes Medical Institute, University of Massachusetts Chan Medical School, Worcester, MA, USA.
Institute of Anatomy, University of Bern, 3012 Bern, Switzerland.
IUCrJ. 2025 Jul 1;12(Pt 4):502-510. doi: 10.1107/S2052252525005196.
Cryo-electron microscopy can be used to image cells and tissue at high resolution. To ensure electron transparency, the sample thickness must not exceed 500 nm. Focused-ion-beam (FIB) milling has become the standard method for preparing thin samples (lamellae); however, the material removed by the milling process is lost, the imageable area is usually limited to a few square micrometres and the surface layers sustain damage from the ion beam. We have examined cryo-electron microscopy of vitreous sections (CEMOVIS), a technique based on cutting thin sections with a knife, as an alternative to FIB milling. Vitreous sections also sustain damage, including compression, shearing and cracks. However, samples can be sectioned in series, producing many orders of magnitude more imageable area compared to lamellae, making CEMOVIS an alternative to FIB milling with distinct advantages. Using two-dimensional template matching on images of vitreous sections of Saccharomyces cerevisiae cells, we reconstructed the 60S ribosomal subunit at near-atomic resolution, demonstrating that, in many regions of the sections, the molecular structure of these subunits is largely intact, comparable to FIB-milled lamellae.
冷冻电子显微镜可用于对细胞和组织进行高分辨率成像。为确保电子透明度，样品厚度不得超过500纳米。聚焦离子束（FIB）铣削已成为制备薄样品（薄片）的标准方法；然而，铣削过程中去除的材料会丢失，可成像区域通常限制在几平方微米，并且表层会受到离子束的损伤。我们研究了玻璃切片冷冻电子显微镜（CEMOVIS），这是一种基于用刀切割薄片的技术，作为FIB铣削的替代方法。玻璃切片也会受到损伤，包括压缩、剪切和裂纹。然而，样品可以连续切片，与薄片相比可成像区域增加了许多个数量级，这使得CEMOVIS成为FIB铣削的一种具有明显优势的替代方法。通过对酿酒酵母细胞玻璃切片图像进行二维模板匹配，我们以近原子分辨率重建了60S核糖体亚基，表明在切片的许多区域，这些亚基的分子结构基本完整，与FIB铣削的薄片相当。
