Chapter 2 - Cryo-FIB developments to enable nanoscale biopsies
- Author(s)
- Rigort, Alexander; Leis, Andrew;
- Abstract
- The advent of cryo-focused ion beam (FIB) technology has led to significant advancements in the field of in situ cryo-electron tomography, enabling the analysis of proteins in their spatial context within cells. The preparation of thin cellular specimens is essential for tomography, as the thickness of specimens for transmission electron microscopy must be considered in terms of the mean free path of electrons. Tomography complements cryo-electron microscopy (cryo-EM) single-particle analysis, which can achieve the highest resolution of cryo-EM techniques to date. Resolution in single-particle analysis is made possible by the thin nature of individual ice-embedded proteins, combined with statistical averaging of structures and suppression of noise. However, the requirement for isolated and purified proteins means that single-particle analysis does not allow conclusions to be drawn about context or interactions with other cellular components. The cryo-FIB microscope produces a beam of high-energy ions that can site specifically ablate sample atoms and molecules, allowing the exposure of subcellular regions of the vitrified sample and the generation of so-called in situ lamellae. The ability to reproducibly produce these cryo-lamellae ultimately led to the breakthrough in the application of cryo-electron tomography to cells. This chapter describes how the use of cryo-FIB technology is contributing to the study of biological complexity and what challenges still lie ahead.
- Publisher
- Academic Press
- Keywords
- Bulk milling; Cryo-EM; Cryo-lamellae; Cryo-tomography; FIB; Focused ion beam; Micromachining; Plasma-FIB; Vitrification
- Research Division(s)
- Structural Biology
- Publisher's Version
- https://doi.org/10.1016/B978-0-443-18829-9.00002-5
- Terms of Use/Rights Notice
- Refer to copyright notice on published article.
Creation Date: 2025-12-05 09:30:39
Last Modified: 2025-12-05 09:30:58