Achieving High-Quality Tomographic Data Collection Using the 200 kV Transmission Electron Microscope with Dual...

Cryo-electron tomography (cryo-ET) has emerged as a powerful technique for elucidating the structures of cellular organelles and large protein complexes in their native environments. Unlike conventional room-temperature electron microscopy, cryo-ET minimizes radiation-induced and preparation-induced damage to internal structures. Moreover, in contrast to single-particle analysis (SPA), cryo-ET reduces the need for repetitive purification, thereby preserving the most authentic structural information of the samples. To achieve high-resolution imaging, cryo-ET also imposes stringent requirements on the imaging conditions of the TEM. Despite the continuous improvement in cryo-ET data acquisition efficiency and the emergence of numerous software platforms, most solutions are proprietary, vendor-specific, and tightly coupled to certain electron microscope models, leading to high implementation costs. In this study, we installed the open-source software SerialEM on a 200 kV electron microscope (Glacios 2 cryo-TEM) with a dual condenser lens system and performed comprehensive calibrations to enable high-quality tomographic data acquisition. Using apoferritin as a model specimen, we describe the entire workflow-from cryo-sample preparation to automated cryo-ET data collection, followed by tomogram reconstruction and sub-tomogram analysis. This method offers a cost-effective and accessible solution for cryo-ET data collection, particularly for laboratories using Glacios microscopes without access to proprietary software.