Abstract
Abstract:Cryo-electron tomography (cryo-ET) enables visualization of protein complexes within their native cellular environment at molecular resolution. Most cells and all tissues, however, are too thick to be imaged directly by transmission electron microscopy (TEM). Overcoming this limitation requires the production of thin biological sections called lamellae. The procedure to obtain lamellae of cells, either seeded or grown directly on electron microscopy grids, requires cryo-focused ion beam (cryo-FIB) milling to thin the samples. This method faces an additional challenge when dealing with tissues and multicellular organisms, as these samples must be high-pressure frozen, which embeds the sample in a thick layer of ice. Nonetheless, lamellae can still be prepared from such samples by extracting a small volume and transferring it to a receiver grid for lamella preparation, a process called lift-out. Here, we describe the available workflows to produce lamellae by lift-out at cryogenic conditions and recent developments in gas injection system (GIS)-free approaches to the lift-out transfer. These advances expand the applications of cryo-ET, enabling the investigation of tissues and whole organisms in situ at molecular resolution.
Highlights
Transmission electron microscopy (TEM) of biological specimens using cryogenic conditions has transformed the way structural biology is done
While high resolution can be achieved with Single particle analysis (SPA), one of the drawbacks is that the purification step erases any spatial and contextual information about how a protein interacts with other proteins in its native cellular environment
Tomography of lamellae prepared by cryo-FIB milling has provided unprecedented insight into the native ultrastructure of unicellular organisms and cells cultured on 2D supports, shedding light on cellular phenomena and molecular complexes, for example, in algae [1], yeast [2], and mammalian cells [3]
Summary
Transmission electron microscopy (TEM) of biological specimens using cryogenic conditions has transformed the way structural biology is done. Tomography of lamellae prepared by cryo-FIB milling has provided unprecedented insight into the native ultrastructure of unicellular organisms and cells cultured on 2D supports, shedding light on cellular phenomena and molecular complexes, for example, in algae [1], yeast [2], and mammalian cells [3]. The complications associated with thicker specimens arise mainly from the cryo-fixation process itself: the water inside the biological specimen needs to be frozen extremely fast to transform it into a non-crystalline amorphous, or glass-like, state This rapid freezing process prevents the formation of ice crystals, which would otherwise damage the cellular ultrastructure. We will introduce advancements in the cryo-lift-out method that facilitate sample preparation of high-pressure-frozen samples
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