ABSTRACT
Rapidly changing features in an intact biological sample are challenging to efficiently trap and image by conventional electron microscopy (EM). For example, the model organism C. elegans is widely used to study embryonic development and differentiation, yet the fast kinetics of cell division makes the targeting of specific developmental stages for ultrastructural study difficult. We set out to image the condensed metaphase chromosomes of an early embryo in the intact worm in 3-D. To achieve this, one must capture this transient structure, then locate and subsequently image the corresponding volume by EM in the appropriate context of the organism, all while minimizing a variety of artifacts. In this methodological advance, we report on the high-pressure freezing of spatially constrained whole C. elegans hermaphrodites in a combination of cryoprotectants to identify embryonic cells in metaphase by in situ cryo-fluorescence microscopy. The screened worms were then freeze substituted, resin embedded and further prepared such that the targeted cells were successfully located and imaged by focused ion beam scanning electron microscopy (FIB-SEM). We reconstructed the targeted metaphase structure and also correlated an intriguing punctate fluorescence signal to a H2B-enriched putative polar body autophagosome in an adjacent cell undergoing telophase. By enabling cryo-fluorescence microscopy of thick samples, our workflow can thus be used to trap and image transient structures in C. elegans or similar organisms in a near-native state, and then reconstruct their corresponding cellular architectures at high resolution and in 3-D by correlative volume EM.
Subject(s)
Caenorhabditis elegans , Imaging, Three-Dimensional , Animals , Cryoelectron Microscopy , Freezing , Microscopy, Electron , Microscopy, Electron, Scanning , Microscopy, FluorescenceABSTRACT
Cryo-electron tomography (cryo-ET) is a groundbreaking technology for 3D visualisation and analysis of biomolecules in the context of cellular structures. It allows structural investigations of single proteins as well as their spatial arrangements within the cell. Cryo-tomograms provide a snapshot of the complex, heterogeneous and transient subcellular environment. Due to the excellent structure preservation in amorphous ice, it is possible to study interactions and spatial relationships of proteins in their native state without interference caused by chemical fixatives or contrasting agents. With the introduction of focused ion beam (FIB) technology, the preparation of cellular samples for electron tomography has become much easier and faster. The latest generation of integrated FIB and scanning electron microscopy (SEM) instruments (dual beam microscopes), specifically designed for cryo-applications, provides advances in automation, imaging and the preparation of high-pressure frozen bulk samples using cryo-lift-out technology. In addition, correlative cryo-fluorescence microscopy provides cellular targeting information through integrated software and hardware interfaces. The rapid advances, based on the combination of correlative cryo-microscopy, cryo-FIB and cryo-ET, have already led to a wealth of new insights into cellular processes and provided new 3D image data of the cell. Here we introduce our recent developments within the cryo-tomography workflow, and we discuss the challenges that lie ahead. LAY DESCRIPTION: This article describes our recent developments for the cryo-electron tomography (cryo-ET) workflow. Cryo-ET offers superior structural preservation and provides 3D snapshots of the interior of vitrified cells at molecular resolution. Before a cellular sample can be imaged by cryo-ET, it must be made accessible for transmission electron microscopy. This is achieved by preparing a 200-300 nm thin cryo-lamella from the cellular sample using a cryo-focused ion beam (cryo-FIB) microscope. Cryo-correlative light and electron microscopy (cryo-CLEM) is used within the workflow to guide the cryo-lamella preparation to the cellular areas of interest. We cover a basic introduction of the cryo-ET workflow and show new developments for cryo-CLEM, which facilitate the connection between the cryo-light microscope and the cryo-FIB. Next, we present our progress in cryo-FIB software automation to streamline cryo-lamella preparation. In the final section we demonstrate how the cryo-FIB can be used for 3D imaging and how bulk-frozen cellular samples (obtained by high-pressure freezing) can be processed using the newly developed cryo-lift-out technology.
Subject(s)
Cryoelectron Microscopy , Electron Microscope Tomography , Automation , Microscopy, Electron, Scanning , Microscopy, Electron, Transmission , WorkflowABSTRACT
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.