Abstract:Cryo-electron tomography (cryo-ET) has the potential to revolutionize our understanding of the building blocks of life since it provides the unique opportunity to study molecules and membrane architectures in the context of cellular interaction. In particular, the combination of fluorescence imaging with focused ion beam (FIB) milling allows the targeting of specific structures in thick cellular samples by preparing thin lamellae that contain a specific fluorescence marker. This technique has conventionally been time-consuming, as it requires sample transfer to multiple microscopes and presents several technical challenges that currently limit its success. Here we describe METEOR, a FIB-integrated microscopy solution that streamlines the correlative cryo-ET workflow. It protects the sample from ice contamination by minimizing handling steps, thus increasing the likelihood of high-quality data. It also allows for monitoring of the milling procedure to ensure the molecule of interest is captured and can then be imaged by cryo-ET.
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.
Cryo-focused ion beam milling of frozen-hydrated cells and subsequent cryo-electron tomography (cryo-ET) has enabled the structural elucidation of macromolecular complexes directly inside cells. Application of the technique to multicellular organisms and tissues, however, is still limited by sample preparation. While high-pressure freezing enables the vitrification of thicker samples, it prolongs subsequent preparation due to increased thinning times and the need for extraction procedures. Additionally, thinning removes large portions of the specimen, restricting the imageable volume to the thickness of the final lamella, typically < 300 nm. Here, we introduce Serial Lift-Out, an enhanced lift-out technique that increases throughput and obtainable contextual information by preparing multiple sections from single transfers. We apply Serial Lift-Out to C. elegans L1 larvae yielding a cryo-ET dataset sampling the worm's anterior-posterior axis and resolve its ribosome structure to 7 A, illustrating how Serial Lift-Out enables the study of multicellular molecular anatomy.
Cryogenic electron tomography (cryo-ET) is emerging as a powerful technique to acquire highresolution 3D structures such as intracellular organelles and protein complexes in their near-native cellular environment. Most cellular samples require thinning through cryo focussed ion beam (FIB) milling to create an electron transparent cell section called a lamella. Cryo-correlative microscopy, which requires acquiring images prior to cryo-FIB milling on a separate cryo-fluorescent light microscope (FLM), has proven an excellent technique to target a specific regions of interest (ROI) [1,2]. However, this sample preparation workflow is laborious, time-consuming, and prone to sample contamination and damage, due to multiple transfer steps. To overcome this limitation, fluorescence imaging systems which are directly integrated in the FIB/SEM chamber have been developed in recent years [3,4].Here we present METEOR, a commercially available integrated widefield FLM that enables correlated cryo-FIB-milling of biological samples in one device. Besides avoiding unnecessary transfer steps it allows the user to verify the presence of the targeted fluorescent signal in lamellae during and after the milling process.We show that METEOR can be used to target ROI's for cryo-FIB milling in a variety of cell types including yeast and HeLa cells. We also present different workflows METEOR can be incorporated into.
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