Methods of enhanced FIB-SEM sample preparation and image acquisition

The ability to view biomolecules in cells and measure changes in their structure, quantity, distribution, and interaction is fundamental to understanding biology. By coupling nano -scale resolution with meso and even macro scale volumes, the enhanced focused ion beam-scanning electron microscopy (FIB-SEM) pipeline has enabled numerous transformational discoveries in life science, many of which were major new landmarks in their fields. This pipeline consists of EM sample preparation, FIB-SEM sample preparation, FIB-SEM imaging, data alignment, and image analysis. While the EM sample preparation, data alignment, and image analysis are consistent with those from other volume Electron Microscopy (vEM) approaches, the enhanced FIB-SEM sample preparation and imaging are unique to the rest of comparable methods. We here illustrate the detailed methods of enhanced FIB-SEM sample preparation and image acquisition that have not been previously described. These methods can also be applied to the conventional FIB-SEM platforms for improved image acquisition quality and pipeline throughput.