Advanced cryo-tomography workflow developments - correlative microscopy, milling automation and cryo-lift-out.

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.

Cryo-focused-ion-beam applications in structural biology.

The ability to precisely control the preparation of biological samples for investigations by electron cryo-microscopy is becoming increasingly important for ultrastructural imaging in biology. Precision machining instruments such as the focused ion beam microscope (FIB) were originally developed for applications in materials science. However, today we witness a growing use of these tools in the life sciences mainly due to their versatility, since they can be used both as manipulation and as imaging devices, when complemented with a scanning electron microscope (SEM). The advent of cryo-preparation equipment and accessories made it possible to pursue work on frozen-hydrated biological specimens with these two beam (FIB/SEM) instruments. In structural biology, the cryo-FIB can be used to site-specifically thin vitrified specimens for transmission electron microscopy (TEM) and tomography. Having control over the specimen thickness is a decisive factor for TEM imaging, as the thickness of the object under scrutiny determines the attainable resolution. Besides its use for TEM preparation, the FIB/SEM microscope can be additionally used to obtain three-dimensional volumetric data from biological specimens. The unique combination of an imaging and precision manipulation tool allows sequentially removing material with the ion beam and imaging the milled block faces by scanning with the electron beam, an approach known as FIB/SEM tomography. This review covers both fields of cryo-FIB applications: specimen preparation for TEM cryo-tomography and volume imaging by cryo-FIB/SEM tomography.

Focused ion beam micromachining of eukaryotic cells for cryoelectron tomography.

Cryoelectron tomography provides unprecedented insights into the macromolecular and supramolecular organization of cells in a close-to-living state. However because of the limited thickness range (< 0.5-1 µm) that is accessible with today's intermediate voltage electron microscopes only small prokaryotic cells or peripheral regions of eukaryotic cells can be examined directly. Key to overcoming this limitation is the ability to prepare sufficiently thin samples. Cryosectioning can be used to prepare thin enough sections but suffers from severe artefacts, such as substantial compression. Here we describe a procedure, based upon focused ion beam (FIB) milling for the preparation of thin (200-500 nm) lamellae from vitrified cells grown on electron microscopy (EM) grids. The self-supporting lamellae are apparently free of distortions or other artefacts and open up large windows into the cell's interior allowing tomographic studies to be performed on any chosen part of the cell. We illustrate the quality of sample preservation with a structure of the nuclear pore complex obtained from a single tomogram.

Automated tracing of filaments in 3D electron tomography reconstructions using Sculptor and Situs.

The molecular graphics program Sculptor and the command-line suite Situs are software packages for the integration of biophysical data across spatial resolution scales. Herein, we provide an overview of recently developed tools relevant to cryo-electron tomography (cryo-ET), with an emphasis on functionality supported by Situs 2.7.1 and Sculptor 2.1.1. We describe a work flow for automatically segmenting filaments in cryo-ET maps including denoising, local normalization, feature detection, and tracing. Tomograms of cellular actin networks exhibit both cross-linked and bundled filament densities. Such filamentous regions in cryo-ET data sets can then be segmented using a stochastic template-based search, VolTrac. The approach combines a genetic algorithm and a bidirectional expansion with a tabu search strategy to localize and characterize filamentous regions. The automated filament segmentation by VolTrac compares well to a manual one performed by expert users, and it allows an efficient and reproducible analysis of large data sets. The software is free, open source, and can be used on Linux, Macintosh or Windows computers.

Automated segmentation of electron tomograms for a quantitative description of actin filament networks.

Cryo-electron tomography allows to visualize individual actin filaments and to describe the three-dimensional organization of actin networks in the context of unperturbed cellular environments. For a quantitative characterization of actin filament networks, the tomograms must be segmented in a reproducible manner. Here, we describe an automated procedure for the segmentation of actin filaments, which combines template matching with a new tracing algorithm. The result is a set of lines, each one representing the central line of a filament. As demonstrated with cryo-tomograms of cellular actin networks, these line sets can be used to characterize filament networks in terms of filament length, orientation, density, stiffness (persistence length), or the occurrence of branching points.

Micromachining tools and correlative approaches for cellular cryo-electron tomography.

A principal limitation of cryo-transmission electron microscopy performed on cells or tissues is the accessible specimen thickness. This is exacerbated in tomography applications, where the aspect ratio (and thus the apparent specimen thickness) changes considerably during specimen tilting. Cryo-ultramicrotomy is the most obvious way of dealing with this problem; however, frozen-hydrated sections suffer from potentially inconsistent compression that cannot be corrected with certainty, and furthermore, yields of sections that satisfy all of the conditions necessary for tomographic imaging are poor. An alternative approach that avoids mechanical deformations is the use of focused ion beam (FIB) instrumentation, where thinning of the frozen-hydrated specimen occurs through the process of sputtering with heavy ions, typically gallium. Here, we use correlative cryo-fluorescence microscopy to navigate large cellular volumes and to localize specific cellular targets. We show that the selected targets in frozen-hydrated specimens can be accessed directly by focused ion beam milling. We also introduce a novel cryo-planing procedure as a method that could facilitate thinning of large areas of vitreous ice prior to cryo-fluorescence, FIB thinning, and cryo-electron tomography.

Cell migration: mechanisms of rear detachment and the formation of migration tracks.

Cell migration is central to many biological and pathological processes, including embryogenesis, tissue repair and regeneration as well as cancer and the inflammatory response. In general, cell migration can be usefully conceptualized as a cyclic process. The initial response of a cell to a migration-promoting agent is to polarize and extend protrusions in the direction of migration. These protrusions can be large, broad lamellipodia or spike-like filopodia, are usually driven by actin polymerization, and are stabilized by adhering to the extracellular matrix (ECM) via transmembrane receptors of the integrin family linked to the actin cytoskeleton. These adhesions serve as traction sites for migration as the cell moves forward over them, and they must be disassembled at the cell rear, allowing it to detach. The mechanisms of rear detachment and the regulatory processes involved are not well understood. The disassembly of adhesions that is required for detachment depends on a coordinated interaction of actin and actin-binding proteins, signaling molecules and effector enzymes including proteases, kinases and phosphatases. Originally, the biochemically regulated processes leading to rear detachment of migrating cells were thought not to be necessarily accompanied by any loss of cell material. However, it has been shown that during rear detachment long tubular extensions, the retracting fibers, are formed and that "membrane ripping" occurs at the cell rear. By this process, a major fraction of integrin-containing cellular material is left behind forming characteristic migration tracks that exactly mark the way a cell has taken.

Release of integrin macroaggregates as a mechanism of rear detachment during keratinocyte migration.

Cell-substrate adhesion can be mediated by the relatively short-lived focal complexes and focal adhesions and by the more stable hemidesmosomes. During cell migration both types of cell-substrate adhesions must be disrupted allowing the cell rear to detach. Rear detachment has been described to be accompanied by membrane ripping and the loss of cellular material in a variety of cell types including fibroblasts and chondrocytes, but also in fast moving cells such as keratinocytes. Here we show that migrating keratinocytes leave behind "migration tracks" of cellular remnants that can be classified due to their size, distribution and molecular composition. Type I macroaggregates appeared as spherical and tubular structures with a diameter of about 50-100 nm that were arranged like "pearls on a string". These structures apparently derived from fragmentation of long tubular extensions, the retracting fibers, at the cell rear and contained high amounts of beta1 integrin and different alpha integrins that are components of fibronectin and laminin receptors in migrating keratinocytes usually found in focal adhesions. Type II macroaggregates were recognized as spherical structures with a diameter of about 30 - 50 nm that were arranged in clusters scattered over the gaps between type I, macroaggregates. In contrast to type I type II macroaggregates contained high amounts of beta4 integrin and seemed to derive from former hemidesmosomes. Both types of macroaggregates were completely membrane covered, impermeable compartments devoid of cytosolic proteins. Our observations strongly support the concept that the release of macroaggregates represents a distinct cellular mechanism of rear detachment based on the loss of adhesive receptors embedded in membrane-covered cellular remnants.

The secretory beta-amyloid precursor protein is a motogen for human epidermal keratinocytes.

Cell migration is known to be triggered by constituents of the extracellular matrix such as fibronectin and by soluble mediators commonly summarized as motogens. Many growth factors such as the epidermal growth factor (EGF) have been shown to act as motogens. Recently, the secretory N-terminal portion of the beta-amyloid precursor protein (sAPP) has been identified as a keratinocyte growth factor. Hence, in this study we analysed whether sAPP stimulates also keratinocyte migration employing the stroboscopic cell motility assay. The migration velocity as well as the frequency of lamellipodia protrusion and ruffle formation were increased about two-fold thus corresponding to the effect of EGF. Using a newly developed beta1-integrin migration track assay we observed that sAPP increased the proportion of migrating keratinocytes and their directional persistence. sAPP appeared to operate synergistically with fibronectin with respect to its motogenic effect. Using a modified Boyden chamber assay we showed that sAPP besides its chemokinetic effect functions as a chemoattractant. Like EGF, sAPP exerted its motogenic effect through the activation of Rac kinase but the receptor for sAPP appears to be distinct. The results suggest that sAPP operates as a motogen in the human epidermis, where it may participate in the regulation of reepithelialization during wound healing.

Cryo-electron tomography: the challenge of doing structural biology in situ.

Electron microscopy played a key role in establishing cell biology as a discipline, by producing fundamental insights into cellular organization and ultrastructure. Many seminal discoveries were made possible by the development of new sample preparation methods and imaging modalities. Recent technical advances include sample vitrification that faithfully preserves molecular structures, three-dimensional imaging by electron tomography, and improved image-processing methods. These new techniques have enabled the extraction of high fidelity structural information and are beginning to reveal the macromolecular organization of unperturbed cellular environments.

Integrative approaches for cellular cryo-electron tomography: correlative imaging and focused ion beam micromachining.

The application of cryo-electron tomography to cells and tissues is commonly referred to as "cellular tomography," and enables visualization of the supramolecular architecture of cells in a near-native state. However, in order to access structural features hidden deep inside cellular volumes, it is necessary to use hybrid techniques to identify and localize features of interest and prepare such regions for subsequent analysis by transmission electron microscopy. We present a workflow that integrates different approaches: (1) correlative cryo-fluorescence microscopy to localize features within frozen-hydrated cells, (2) focused ion beam milling to thin these specimens in a targeted manner, and (3) cryo-electron tomography to provide detailed information about the cellular ultrastructure of thinned samples. We describe the combined use of these techniques and the instrumentation required to enable cryo-electron tomography for a vast range of cellular samples.

Correlative cryo-light microscopy and cryo-electron tomography: from cellular territories to molecular landscapes.

In cell biology, visual techniques such as light and electron microscopy provide the most intuitive means by which to study structure and function; however, no single microscopy technique is capable of providing all of the desired information. As a consequence, many separate techniques have evolved, each with unique capabilities. The most informative approaches are global in the sense that they take advantage of multiple imaging modalities spanning a range of spatial scales and frequencies, preferably encompassing preservation of the hydrated nature of the cell. Correlative microscopy utilizes complementary visual techniques that allow the experimenter to capture significant proportions of a population of cells, to identify features of interest, and to then capture high-resolution snapshots that represent bona fide cellular events.

Correlative microscopy: bridging the gap between fluorescence light microscopy and cryo-electron tomography.

Cryo-electron tomography of frozen-hydrated biological samples offers a means of studying large and complex cellular structures in three-dimensions and with nanometer-scale resolution. The low contrast of unstained biological material embedded in amorphous ice and the need to minimise the exposure of these radiation-sensitive samples to the electron beam result in a poor signal-to-noise ratio. This poses problems not only in the visualisation and interpretation of such tomograms, it is also a problem in surveying the sample and in finding regions which contain the features of interest and which are suitable for recording tomograms. To address this problem, we have developed a correlative fluorescence light microscopy-electron microscopy approach, which guides the search for the structures of interest and allows electron microscopy to zoom in on them. With our approach, the total dose spent on locating regions of interest is negligible. A newly designed cryo-holder allows imaging of fluorescently labelled samples after vitrification. The absolute coordinates of structures identified and located by cryo-light microscopy are transferred to the electron microscope via a Matlab-based user interface. We have successfully tested the experimental setup and the whole procedure with two types of adherent fluorescently labelled cells, a neuronal cell line and keratinocytes, both grown directly on EM grids.

Structural and compositional analysis of the keratinocyte migration track.

Slowly migrating cells such as fibroblasts leave behind a "migration track," which has been assumed not to occur in fast-moving cells such as keratinocytes. Here we show that keratinocytes left behind "migration tracks" of cellular remnants consisting of membranous patches or macroaggregates that were anchored to a meshwork of extracellular matrix proteins consisting of collagen type IV, fibronectin, laminin, and laminin 5. According to their origin and localisation, two types of macroaggregates could be distinguished : (1) Spherical and elongated tubular structures (diameter about 50-110 nm) both of which were arranged like "pearls on a string" and that apparently derived from fragmentation of retracting fibres. (2) Spherical structures (diameter about 50 nm) left behind in the gaps between the retracting fibres and presumably derived from former focal adhesion sites. Both types of macroaggregates did not contain cytoplasmic proteins but carried on their surface adhesion proteins, particularly high amounts of integrins : type 1 macroaggregates contained alpha3beta1-integrins, whereas type 2 macroaggregates contained other types of integrins such as alpha6beta4-integrins. Modulation of keratinocyte adhesion by using poly-L-lysine coated cover slips resulted in an increased application of inhibitory beta1-antibodies and slightly reduced migration velocity and track formation. Within 24 h of migration, we observed a migration velocity-dependent loss of cellular beta1-integrin by macroaggregate formation of about 11% for fast and about 4% for slowly migrating keratinocytes. The physiological role of the migration track is unclear. However, with its multiple adhesion sites it may serve as a provisional basement membrane during reepithelialization of epidermal wounds.