Cryo-liftout system for preparing in situ lamellae in cryo-FIBSEM

概述。我们的生物矿化研究中存在着几个主要的挑战，目前迫切需要一种低温升降（cryoLO） FIB-SEM纳米机械手：i)在标本中保存连续的三维信息，ii)在硬生物标本中精确地导航从微米到Ångstroms的尺寸尺度，以观察孤立发现的相关背景下的层次结构，以及iii)获得原始水合状态下有机和无机界面关系的高分辨率信息。结合水在生物矿化过程中是必不可少的，它是生物分子和矿物表面之间界面的结构成分。水在生物系统中很重要，因为它在很大程度上决定了它们的生物力学特性，但在常规样品制备和电子显微镜的高真空环境中很容易丢失。

Overview. Several major challenges exist in our biomineralization research for which a cryo-liftout (cryoLO) FIB-SEM nanomanipulator is urgently needed at this time: i) preserving the continuum of 3D information in specimens, ii) precisely navigating size scales from microns to Ångstroms within hard biospecimens to observe hierarchical structure with correlation context of isolated findings, and iii) obtaining high-resolution information on organic and inorganic interfacial relationships in their pristine hydrated state. Bound water is essential in biomineralization processes, where it is a structural component at the interface between biomolecules and mineral surfaces. Water is important in biological systems because it largely defines their biomechanical properties, but is easily lost during conventional sample preparation and in the high-vacuum environment of electron microscopes. Need/Urgency. Given that both the mineral and organics in our samples are hydrated, with interfacial interactions occurring at the nanoscale in the presence of water, a pressing need is to apply cryo-preparation/imaging technology to retain structural water. A cryoLO is urgently needed to fill a significant gap in our correlative cryo-sample preparation. It addresses the challenges listed above, and will keep us internationally competitive in describing structure and function at the nanoscale in our biomineralization research. We have reached a ceiling using conventional electron microscopy methods, and we urgently need the ability to "go fully cryo" for resolving true, hydrated nanostructure. Our students must receive and apply this cryo-training as they develop their careers in a discipline realizing that cryo is the essential and future way to examine biomineralized structure. Suitability. We already have world-leading research programs using advanced ultrastructural approaches to study biomineralized structures using core instrumentation in McGill's Facility for Electron Microscopy Research (FEMR). Impact. A cryoLO nanomanipulator for our existing dual-beam FIB-SEM electron microscope; McKee CFI-LEF #30797) will enable a unique capability (first in Canada) for describing tomographic detail of hydrated biomineralized structure. With this information as acquired by our planned cryo-workflow using the requested cryoLO, we will understand more broadly and comprehensively, and in the pristine hydrated state at the nanoscale, how mineralized tissues/structures and synthesized biomineral assemblies form and are organized hierarchically to provide their exquisitely tuned mechanical functions. This will keep us at the leading edge for nanostructural characterization of biominerals. In addition to facilitating the research of the applicants, it will have added value in providing much-needed infrastructure for the many other structural biologists that access the FEMR whose research would benefit greatly from cryo-examination of their specimens in a pristine state.