Low temperature FESEM of the calcifying interface of a scleractinian coral

The ultrastructural nature of the calcifying interface in the scleractinian coral Galaxea fascicularis has been investigated using high-resolution, low temperature field emission scanning electron microscopy (FESEM). This technique permitted structural analyses of soft tissue and skeleton in G. fascicularis in a frozen-hydrated state, without the need for chemical fixation or decalcification. Structural comparisons are made between frozen-hydrated polyps and polyps that have undergone conventional fixation and decalcification. Vesicles expelled by the calicoblastic ectodermal cells into sub-skeletal spaces and previously suggested to play a role in calcification were commonly observed in fixed samples but were distinctly absent in frozen-hydrated preparations. We propose that these vesicles are fixation artefacts. Two distinct types of vesicles (380 and 70 nm in diameter, respectively), were predominant throughout the calicoblastic ectodermal cells of frozen-hydrated preparations, but these were never seen to be entering, or to be contained within, sub-skeletal spaces, nor did they contain any crystalline material. In frozen-hydrated preparations, membranous sheets were seen to surround and isolate portions of aboral mesogloea and to form junctional complexes with calicoblastic cells. The calicoblastic ectoderm was closely associated with the underlying skeleton, with sub-skeletal spaces significantly smaller (P<0.0001) in frozen-hydrated polyps compared to fixed polyps. A network of organic filaments (26 nm in diameter) extended from the apical membranes of calicoblastic cells into these small sub-skeletal cavities. A thin sheath was also frequently observed adjacent to the apical membrane of calicoblastic cells.     

Electron tomography: towards visualizing the molecular organization of the cytoplasm

Electron tomography of thin sections of plastic-embedded biological material provides valuable insights into the organellar organization of cells. With the advent of automated data-acquisition procedures, it has become possible to study radiation-sensitive specimens, such as frozen-hydrated organelles and whole cells. This opens up exciting perspectives for visualizing the molecular organization of the cytoplasm.     

Cryo-electron microscopy of cell division in Staphylococcus aureus reveals a mid-zone between nascent cross walls

Cryo-electron microscopy of frozen-hydrated thin sections permits the observation of the real distribution of mass in biological specimens allowing the native structure of bacteria to be seen, including the natural orientation of their surface layers. Here, we use this approach to study the fine ultrastructure of the division site, or septum, of Staphylococcus aureus D₂C. Frozen-hydrated sections revealed a differentiated cell wall at the septum, showing two high-density regions sandwiched between three low-density zones. The two zones adjacent to the membrane appeared as an extension of the periplasmic space seen in this organism's cell envelope and showed no distinguishing structures within them. Immediately next to these were higher-density zones that corresponded to nascent cross walls of the septum. Unexpectedly, a rather broad low-density zone was seen separating cross walls in the septum. This mid-zone of low density appeared inflated and without visible structures in isolated cell walls, which showed only the high-density zones of the septum. Here, we suggest that frozen-hydrated thin sections have captured a highly fragile septal region, the mid-zone, which results from the dynamic action of autolysis and actively separates daughter cells during division. The two zones next to the membranes are periplasmic spaces. Immediately next to these are the growing cross walls composed of peptidoglycan, teichoic acid and protein.     

On the freezing and identification of lipid monolayer 2-D arrays for cryoelectron microscopy

Lipid monolayers provide a convenient vehicle for the crystallization of biological macromolecules for 3-D electron microscopy. Although numerous examples of 3-D images from 2-D protein arrays have been described from negatively stained specimens, only six structures have been done from frozen-hydrated specimens. We describe here a method that makes high quality frozen-hydrated specimens of lipid monolayer arrays for cryoelectron microscopy. The method uses holey carbon films with patterned holes for monolayer recovery, blotting and plunge freezing to produce thin aqueous films which cover >90% of the available grid area. With this method, even specimens with relatively infrequent crystals can be screened using automated data collection techniques. Though developed for microscopic examination of 2-D arrays, the method may have wider application to the preparation of single particle specimens for 3-D image reconstruction.     

Towards high-resolution three-dimensional imaging of native mammalian tissue: electron tomography of frozen-hydrated rat liver sections

Cryo-electron tomography of frozen-hydrated specimens holds considerable promise for high-resolution three-dimensional imaging of organelles and macromolecular complexes in their native cellular environment. While the technique has been successfully used with small, plunge-frozen cells and organelles, application to bulk mammalian tissue has proven to be difficult. We report progress with cryo-electron tomography of frozen-hydrated sections of rat liver prepared by high-pressure freezing and cryo-ultramicrotomy. Improvements include identification of suitable grids for mounting sections for tomography, reduction of surface artifacts on the sections, improved image quality by the use of energy filtering, and more rapid tissue excision using a biopsy needle. Tomographic reconstructions of frozen-hydrated liver sections reveal the native structure of such cellular components as mitochondria, endoplasmic reticulum, and ribosomes, without the selective attenuation or enhancement of ultrastructural details associated with the osmication and post-staining used with freeze-substitution.     

Three-dimensional reconstruction of caldesmon-containing smooth muscle thin filaments

Caldesmon is known to inhibit actomyosin ATPase and filament sliding in vitro, and may play a role in modulating smooth muscle contraction as well as in diverse cellular processes including cytokinesis and exocytosis. However, the structural basis of caldesmon action has not previously been apparent. We have recorded electron microscope images of negatively stained thin filaments containing caldesmon and tropomyosin which were isolated from chicken gizzard smooth muscle in EGTA. Three-dimensional helical reconstructions of these filaments show actin monomers whose bilobed shape and connectivity are very similar to those previously seen in reconstructions of frozen-hydrated skeletal muscle thin filaments. In addition, a continuous thin strand of density follows the long-pitch actin helices, in contact with the inner domain of each actin monomer. Gizzard thin filaments treated with Ca2+/calmodulin, which dissociated caldesmon but not tropomyosin, have also been reconstructed. Under these conditions, reconstructions also reveal a bilobed actin monomer, as well as a continuous surface strand that appears to have moved to a position closer to the outer domain of actin. The strands seen in both EGTA- and Ca2+/calmodulin-treated filaments thus presumably represent tropomyosin. It appears that caldesmon can fix tropomyosin in a particular position on actin in the absence of calcium. An influence of caldesmon on tropomyosin position might, in principle, account for caldesmon's ability to modulate actomyosin interaction in both smooth muscles and non-muscle cells.     

Projected structure of the pore-forming OmpC protein from Escherichia coli outer membrane.

A single-projection structure analysis of a bacterial outer membrane protein, OmpC, has been carried out by electron microscopy of frozen hydrated specimens. Two distinct crystal polymorphs have been observed in the frozen-hydrated samples, and projection structures of both forms have been obtained to a resolution of 13.5 A. Preliminary examination of negatively stained samples revealed the expected, trimeric appearance of pores in the OmpC specimens. Electron microscopy of unstained, frozen-hydrated OmpC reveals the trimeric pore structure with equal clarity. In addition, the overall molecular envelope of the protein is readily discerned, and a major lipid-containing domain can also be seen. Because of the small coherent patch size, mosaic disorder, and unpredictable polymorphism of the presently available specimens, three-dimensional reconstruction of frozen-hydrated OmpC has not been carried out.     

Preparation and analysis of large, flat crystals of Ca(2+)-ATPase for electron crystallography.

Obtaining large, flat, well ordered crystals represents the key to structure determination by electron crystallography. Multilamellar crystals of Ca(2+)-ATPase are a good candidate for this methodology, and we have optimized methods of crystallization and of preparation for cryoelectron microscopy. In particular, high concentrations of glycerol were found to prevent nucleation and to reduce stacking; thus, by seeding solutions containing 40% glycerol, we obtained thin crystals that were 5-30 microns in diameter and 2-10 unit cells thick. We found that removing vesicles and minimizing concentrations of divalent cations were critical to preparing flat crystals in the frozen-hydrated state. Finally, we developed two methods for determining the number of lamellae composing individual crystals, information that is required for structure determination of this crystal form. The first method, using low magnification images of freeze-dried crystals, is more practical in our case. Nevertheless, the alternative method, involving analysis of Laue zones from electron diffraction patterns of slightly tilted crystals, may be of general use in structure determination from thin, three-dimensional crystals.     

Direct imaging of polysaccharide aggregates in frozen aqueous dilute systems

The frozen-hydrated/cryo-transmission electron microscopy technique has been used to observe polysaccharide aggregates in vitreous ice. Images of kappacarrageenan in the helical state showed fairly large aggregates of a loosely intertwined network of thin rigid rods. Aggregates of agarose in the helical conformation were also visualised. In comparison with those of kappa-carrageenan, the strands of agarose were shorter and less rigid. This difference is discussed in terms of the current knowledge of the gelation of these polysaccharides.     

Cryoelectron microscopy of frozen-hydrated alpha-ketoacid dehydrogenase complexes from Escherichia coli

The native architectures of the pyruvate and 2-oxoglutarate dehydrogenase complexes have been investigated by cryoelectron microscopy of unstained, frozen-hydrated specimens. In pyruvate dehydrogenase complex and 2-oxoglutarate dehydrogenase complex the transacylase (E2) components exist as 24-subunit, cube-shaped assemblies that form the structural cores of the complexes. Multiple copies (12-24) of the alpha-ketoacid dehydrogenase (E1) and dihydrolipoyl dehydrogenase (E3) components bind to the surface of the cores. Images of the frozen-hydrated enzyme complexes do not appear consistent with a symmetric arrangement of the E1 and E3 subunits about the octahedrally symmetric E2 core. Often the E1 or E3 subunits appear separated from the surface of the E2 core by 3-5 nm, and sometimes thin bridges of density appear in the gap between the E2 core and the bound subunits; studies of subcomplexes consisting of the E2 core from 2-oxoglutarate dehydrogenase complex and E1 or E3 show that both E1 and E3 are bound in this manner. Images of the E2 cores isolated from pyruvate dehydrogenase complex appear surrounded by a faint fuzz that extends approximately 10 nm from the surface of the core and likely corresponds to the lipoyl domains of the E2.     

Ultrastructure and evolution of ballistosporic basidiospores

Basidiospores oiCoprinus cinereus were examined before and during spore discharge using light microscopy, and SEM of frozen-hydrated and other preparations. The process of droplet development at the hilar appendix was divided into three stages: pre-droplet, early and late enlargement. The fully expanded droplet was preserved only in frozen-hydrated specimens. Two-step droplet enlargement was also observed with TEM in Boletus rubinellus, and the droplet at the early enlargement stage was enclosed by a trilaminate membrane. The droplet was not observed in Auricularia species, but basidiospore preservation for SEM was optimum in Auricularia auricula with frozen-hydrated preparations. The hilar appendix of Auricularia fuscosuccinea as studied by TEM was simple compared with those of the Homobasidiomycetes. The implications of the data for evolution of the ballistosporic basidiospore and the discharge mechanism are considered.     

Interactions between actin and myosin filaments in skeletal muscle visualized in frozen-hydrated thin sections.

For the purpose of determining net interactions between actin and myosin filaments in muscle cells, perhaps the single most informative view of the myofilament lattice is its averaged axial projection. We have studied frozen-hydrated transverse thin sections with the goal of obtaining axial projections that are not subject to the limitations of conventional thin sectioning (suspect preservation of native structure) or of equatorial x-ray diffraction analysis (lack of experimental phases). In principle, good preservation of native structure may be achieved with fast freezing, followed by low-dose electron imaging of unstained vitrified cryosections. In practice, however, cryosections undergo large-scale distortions, including irreversible compression; furthermore, phase contrast imaging results in a nonlinear relationship between the projected density of the specimen and the optical density of the micrograph. To overcome these limitations, we have devised methods of image restoration and generalized correlation averaging, and applied them to cryosections of rabbit psoas fibers in both the relaxed and rigor states. Thus visualized, myosin filaments appear thicker than actin filaments by a much smaller margin than in conventional thin sections, and particularly so for rigor muscle. This may result from a significant fraction of the myosin S1-cross-bridges averaging out in projection and thus contributing only to the baseline of projected density. Entering rigor incurs a loss of density from an annulus around the myosin filament, with a compensating accumulation of density around the actin filament. This redistribution of mass represents attachment of the fraction of cross-bridges that are visible above background. Myosin filaments in the "nonoverlap" zone appear to broaden on entering rigor, suggesting that on deprivation of ATP, cross-bridges in situ move outwards even without actin in their immediate proximity.     

Electron microscopy and computer image averaging of ice-embedded large ribosomal subunits from Escherichia coli

Electron micrographs of frozen-hydrated, large ribosomal subunits from Escherichia coli have been analyzed by computer image processing. Images of subunits in the so-called "crown" orientation were analyzed by correlation alignment procedures developed for negatively stained specimens. Averages of the aligned images showed both similarities and differences to averages determined for negatively stained specimens. The L1 ridge is more dense and stalk-like in frozen-hydrated as compared with negatively stained subunits, possibly because it is associated with ribosomal RNA. The results show that it should be feasible to determine the three-dimensional structure of the large ribosomal subunit from micrographs of individual, frozen-hydrated subunits that have been tilted in the electron microscope.     

Probing the structure and function of the nuclear pore complex.

Nucleocytoplasmic transport is a bi-directional process mediated by the nuclear pore complex (NPC), which results in a segregation of cytoplasmic and nuclear macromolecules within cells. Some progress has been made in understanding the mechanistic basis of this selective transport phenomenon. In particular, cryo-electron microscopy of frozen-hydrated nuclear envelopes coupled with image processing and labeling studies, has provided a glimpse of the transporter at the center of the NPC.     

Electron tomography of cells

The electron microscope has contributed deep insights into biological structure since its invention nearly 80 years ago. Advances in instrumentation and methodology in recent decades have now enabled electron tomography to become the highest resolution three-dimensional (3D) imaging technique available for unique objects such as cells. Cells can be imaged either plastic-embedded or frozen-hydrated. Then the series of projection images are aligned and back-projected to generate a 3D reconstruction or 'tomogram'. Here, we review how electron tomography has begun to reveal the molecular organization of cells and how the existing and upcoming technologies promise even greater insights into structural cell biology.     

Radiation dose reduction and image enhancement in biological imaging through equally-sloped tomography

Electron tomography is currently the highest resolution imaging modality available to study the 3D structures of pleomorphic macromolecular assemblies, viruses, organelles and cells. Unfortunately, the resolution is currently limited to 3-5nm by several factors including the dose tolerance of biological specimens and the inaccessibility of certain tilt angles. Here we report the first experimental demonstration of equally-sloped tomography (EST) to alleviate these problems. As a proof of principle, we applied EST to reconstructing frozen-hydrated keyhole limpet hemocyanin molecules from a tilt-series taken with constant slope increments. In comparison with weighted back-projection (WBP), the algebraic reconstruction technique (ART) and the simultaneous algebraic reconstruction technique (SART), EST reconstructions exhibited higher contrast, less peripheral noise, more easily detectable molecular boundaries and reduced missing wedge effects. More importantly, EST reconstructions including only two-thirds the original images appeared to have the same resolution as full WBP reconstructions, suggesting that EST can either reduce the dose required to reach a given resolution or allow higher resolutions to be achieved with a given dose. EST was also applied to reconstructing a frozen-hydrated bacterial cell from a tilt-series taken with constant angular increments. The results confirmed similar benefits when standard tilts are utilized.     

Structure of CaATPase: electron microscopy of frozen-hydrated crystals at 6 A resolution in projection

Thin, three-dimensional crystals of CaATPase have been studied at high resolution by electron crystallography. These crystals were grown by adding purified CaATPase to appropriate concentrations of lipid, detergent and calcium. A thin film of crystals was then rapidly frozen and maintained in the frozen-hydrated state during electron microscopy. The resulting electron diffraction patterns extend to 4.1 A resolution and images contain phase data to 6 A resolution. By combining Fourier amplitudes from electron diffraction patterns with phases from images, a density map has been calculated in projection. Comparison of this map from unstained crystals with a previously determined map from negatively stained crystals reveals distinct contributions from intramembranous and extramembranous protein domains. On the basis of this distinction and of the packing of molecules in the crystal, we have proposed a specific arrangement for the ten alpha-helices that have been suggested as spanning the bilayer.     

Beam-induced motion of vitrified specimen on holey carbon film

The contrast observed in images of frozen-hydrated biological specimens prepared for electron cryo-microscopy falls significantly short of theoretical predictions. In addition to limits imposed by the current instrumentation, it is widely acknowledged that motion of the specimen during its exposure to the electron beam leads to significant blurring in the recorded images. We have studied the amount and direction of motion of virus particles suspended in thin vitrified ice layers across holes in perforated carbon films using exposure series. Our data show that the particle motion is correlated within patches of 0.3-0.5 μm, indicating that the whole ice layer is moving in a drum-like motion, with accompanying particle rotations of up to a few degrees. Support films with smaller holes, as well as lower electron dose rates tend to reduce beam-induced specimen motion, consistent with a mechanical effect. Finally, analysis of movies showing changes in the specimen during beam exposure show that the specimen moves significantly more at the start of an exposure than towards its end. We show how alignment and averaging of movie frames can be used to restore high-resolution detail in images affected by beam-induced motion.     

Three-dimensional cellular ultrastructure resolved by X-ray microscopy

We developed an X-ray microscope using partially coherent object illumination instead of previously used quasi-incoherent illumination. The design permitted the incorporation of a cryogenic tilt stage, enabling tomography of frozen-hydrated, intact adherent cells. We obtained three-dimensional reconstructions of mouse adenocarcinoma cells at ～36-nm (Rayleigh) and ～70-nm (Fourier ring correlation) resolution, which allowed us to visualize the double nuclear membrane, nuclear pores, nuclear membrane channels, mitochondrial cristae and lysosomal inclusions.     

Preparation of macromolecular complexes for cryo-electron microscopy

This protocol describes the preparation of frozen-hydrated single-particle specimens of macromolecular complexes. First, it describes how to create a grid surface coated with holey carbon by first inducing holes in a Formvar film to act as a template for the holey carbon that is stable under cryo-electron microscopy (cryo-EM) conditions and is sample-friendly. The protocol then describes the steps required to deposit the homogeneous sample on the grid and to plunge-freeze the grid into liquid ethane at the temperature of liquid nitrogen, so that it is suitable for cryo-EM visualization. It takes 4-5 h to make several hundred holey carbon grids and about 1 h to make the frozen-hydrated grids. The time required for sample purification varies from hours to days, depending on the sample and the specific procedure required. A companion protocol details how to collect cryo-EM data using an FEI Tecnai transmission electron microscope that can subsequently be processed to obtain a three-dimensional reconstruction of the macromolecular complex.