Project description:Single particle tomography (SPT), also known as subtomogram averaging, is a powerful technique uniquely poised to address questions in structural biology that are not amenable to more traditional approaches like X-ray crystallography, nuclear magnetic resonance, and conventional cryoEM single particle analysis. Owing to its potential for in situ structural biology at subnanometer resolution, SPT has been gaining enormous momentum in the last five years and is becoming a prominent, widely used technique. This method can be applied to unambiguously determine the structures of macromolecular complexes that exhibit compositional and conformational heterogeneity, both in vitro and in situ. Here we review the development of SPT, highlighting its applications and identifying areas of ongoing development.
Project description:Micro-electron diffraction (MicroED) is an emerging technique to use cryo-electron microscope to study the crystal structures of macromolecule from its micro-/nano-crystals, which are not suitable for conventional X-ray crystallography. However, this technique has been prevented for its wide application by the limited availability of producing good micro-/nano-crystals and the inappropriate transfer of crystals. Here, we developed a complete workflow to prepare suitable crystals efficiently for MicroED experiment. This workflow includes in situ on-grid crystallization, single-side blotting, cryo-focus ion beam (cryo-FIB) fabrication, and cryo-electron diffraction of crystal cryo-lamella. This workflow enables us to apply MicroED to study many small macromolecular crystals with the size of 2-10 ?m, which is too large for MicroED but quite small for conventional X-ray crystallography. We have applied this method to solve 2.5 Å crystal structure of lysozyme from its micro-crystal within the size of 10?×?10?×?10 ?m3. Our work will greatly expand the availability space of crystals suitable for MicroED and fill up the gap between MicroED and X-ray crystallography.
Project description:Imaging of cells and tissues has improved significantly over the last decade. Dual-beam instruments with a focused ion beam mounted on a scanning electron microscope (FIB-SEM), offering high-resolution 3D imaging of large volumes and fields-of-view are becoming widely used in the life sciences. FIB-SEM has most recently been implemented on fully hydrated, cryo-immobilized, biological samples. Correlative light and electron microscopy workflows combining fluorescence microscopy (FM) with FIB-SEM imaging exist, whereas workflows combining cryo-FM and cryo-FIB-SEM imaging are not yet commonly available. Here, we demonstrate that fluorescently labeled lipid droplets can serve as in situ fiducial markers for correlating cryo-FM and FIB-SEM datasets and that this approach can be used to target the acquisition of large FIB-SEM stacks spanning tens of microns under cryogenic conditions. We also show that cryo-FIB-SEM imaging is particularly informative for questions related to organelle structure and inter-organellar contacts, nuclear organization, and mineral deposits in cells.
Project description:Studying bacterial cell envelope architecture with electron microscopy is challenging due to the poor preservation of microbial ultrastructure with traditional methods. Here, we established and validated a super-resolution cryo-correlative light and electron microscopy (cryo-CLEM) method, and combined it with cryo-focused ion beam (cryo-FIB) milling and scanning electron microscopy (SEM) volume imaging to structurally characterize the bacterium Deinococcus radiodurans. Subsequent cryo-electron tomography (cryo-ET) revealed an unusual diderm cell envelope architecture with a thick layer of peptidoglycan (PG) between the inner and outer membranes, an additional periplasmic layer, and a proteinaceous surface S-layer. Cells grew in tetrads, and division septa were formed by invagination of the inner membrane (IM), followed by a thick layer of PG. Cytoskeletal filaments, FtsA and FtsZ, were observed at the leading edges of constricting septa. Numerous macromolecular complexes were found associated with the cytoplasmic side of the IM. Altogether, our study revealed several unique ultrastructural features of D. radiodurans cells, opening new lines of investigation into the physiology and evolution of the bacterium.
Project description:Cryo-electron tomography (cryo-ET) is a well-established technique for studying 3D structural details of subcellular macromolecular complexes and organelles in their nearly native context in the cell. A primary limitation of the application of cryo-ET is the accessible specimen thickness, which is less than the diameters of almost all eukaryotic cells. It has been shown that focused ion beam (FIB) milling can be used to prepare thin, distortion-free lamellae of frozen biological material for high-resolution cryo-ET. Commercial cryosystems are available for cryo-FIB specimen preparation, however re-engineering and additional fixtures are often essential for reliable results with a particular cryo-FIB and cryo-transmission electron microscope (cryo-TEM). Here, we describe our optimized protocol and modified instrumentation for cryo-FIB milling to produce thin lamellae and subsequent damage-free cryotransfer of the lamellae into our cartridge-type cryo-TEM.
Project description:Lamella micromachining by focused ion beam milling at cryogenic temperature (cryo-FIB) has matured into a preparation method widely used for cellular cryo-electron tomography. Due to the limited ablation rates of low Ga+ ion beam currents required to maintain the structural integrity of vitreous specimens, common preparation protocols are time-consuming and labor intensive. The improved stability of new-generation cryo-FIB instruments now enables automated operations. Here, we present an open-source software tool, SerialFIB, for creating automated and customizable cryo-FIB preparation protocols. The software encompasses a graphical user interface for easy execution of routine lamellae preparations, a scripting module compatible with available Python packages, and interfaces with three-dimensional correlative light and electron microscopy (CLEM) tools. SerialFIB enables the streamlining of advanced cryo-FIB protocols such as multi-modal imaging, CLEM-guided lamella preparation and in situ lamella lift-out procedures. Our software therefore provides a foundation for further development of advanced cryogenic imaging and sample preparation protocols.
Project description:Cryo-electron tomography (cryo-ET) has enabled high resolution three-dimensional(3D) structural analysis of virus and host cell interactions and many cell signaling events; these studies, however, have largely been limited to very thin, peripheral regions of eukaryotic cells or to small prokaryotic cells. Recent efforts to make thin, vitreous sections using cryo-ultramicrotomy have been successful, however,this method is technically very challenging and with many artifacts. Here, we report a simple and robust method for creating in situ, frozen-hydrated cell lamellas using a focused ion beam at cryogenic temperature (cryo-FIB), allowing access to any interior cellular regions of interest. We demonstrate the utility of cryo-FIB with high resolution 3D cellular structures from both bacterial cells and large mammalian cells. The method will not only facilitate high-throughput 3D structural analysis of biological specimens, but is also broadly applicable to sample preparation of thin films and surface materials without the need for FIB "lift-out".
Project description:Cryo-electron tomography (cryo-ET) is emerging as a revolutionary method for resolving the structure of macromolecular complexes in situ. However, sample preparation for in situ Cryo-ET is labour-intensive and can require both cryo-lamella preparation through cryo-focused ion beam (FIB) milling and correlative light microscopy to ensure that the event of interest is present in the lamella. Here, we present an integrated cryo-FIB and light microscope setup called the Photon Ion Electron microscope (PIE-scope) that enables direct and rapid isolation of cellular regions containing protein complexes of interest. Specifically, we demonstrate the versatility of PIE-scope by preparing targeted cryo-lamellae from subcellular compartments of neurons from transgenic Caenorhabditis elegans and Drosophila melanogaster expressing fluorescent proteins. We designed PIE-scope to enable retrofitting of existing microscopes, which will increase the throughput and accuracy on projects requiring correlative microscopy to target protein complexes. This new approach will make cryo-correlative workflow safer and more accessible.