Abstract
Cryo-electron tomography (cryoET) is a powerful technique that enables the direct study of the molecular structure of tissues and cells. Cryo-focused ion beam (cryoFIB) milling plays an important role in preparation of high-quality thin lamellar samples for cryoET studies, promoting the rapid development of cryoET in recent years. However, locating the regions of interest in a large cell or tissue during cryoFIB milling remains a major challenge limiting cryoET applications on arbitrary biological samples. Here, we report an on-the-fly location method based on cellular secondary electron imaging (CSEI). CSEI is derived from a basic imaging function of the cryoFIB instruments and enables high-contrast imaging of the cellular contents of frozen hydrated biological samples, highlighted by that both fluorescent labels and additional devices are not required. The present work discusses the imaging principles and settings for optimizing CSEI. Tests on several commercially available cryoFIB instruments demonstrated that CSEI was feasible on mainstream instruments to observe all types of cellular contents and was reliable under different milling conditions. Assisted by CSEI, we established a simple milling-location workflow and tested it using the basal body of Chlamydomonas reinhardtii.
Competing Interest Statement
The authors have declared no competing interest.
