On the feasibility of visualizing ultrasmall gold labels in biological specimens by STEM tomography

Labeling with heavy atom clusters attached to antibody fragments is an attractive technique for determining the 3D distribution of specific proteins in cells using electron tomography. However, the small size of the labels makes them very difficult to detect by conventional bright-field electron tomography. Here, we evaluate quantitative scanning transmission electron microscopy (STEM) at a beam voltage of 300 kV for detecting 11-gold atom clusters (Undecagold) and 1.4 nm-diameter nanoparticles (Nanogold) for a variety of specimens and imaging conditions. STEM images as well as tomographic tilt series are simulated by means of the NIST Elastic-Scattering Cross-Section Database for gold clusters embedded in carbon. The simulations indicate that the visibility in 2D of Undecagold clusters in a homogeneous matrix is maximized for low inner collection semi-angles of the STEM annular dark-field detector (15–20 mrad). Furthermore, our calculations show that the visibility of Undecagold in 3D reconstructions is significantly higher than in 2D images for an inhomogeneous matrix corresponding to fluctuations in local density. The measurements demonstrate that it is possible to detect Nanogold particles in plastic sections of tissue freeze-substituted in the presence of osmium. STEM tomography has the potential to localize specific proteins in permeabilized cells using antibody fragments tagged with small heavy atom clusters. Our quantitative analysis provides a framework for determining the detection limits and optimal experimental conditions for localizing these small clusters.     

Ultrastructural localization and identification ofAzospirillum brasilense Cd on and within wheat root by immuno-gold labeling

Azospirillum brasilense Cd localization in wheat roots was studied by light microscopy, by scanning, and by transmission electron microscopy.A. brasilense Cd cells were specifically identified immunocytochemically around and within root tissues.A. brasilense Cd cells found both outside and inside inoculated roots were intensively labeled with colloidal gold. In non-axenic cultures other bacterial strains or plant tissue were not labeled, thereby providing a non-interfering background. The roots of axenic grown wheat plants were colonized both externally and internally byA. brasilense Cd after inoculation, whereas non-axenic cultures were colonized by other bacterial strains as well.A. brasilense Cd cells were located on the root surface along the following zones: the root tip, the elongation, and the root-hair zone. However, bacteria were located within the cortex only in the latter two zones. In a number of observations, an electron dense material mediated the binding of bacterial cells to outer surfaces of epidermal cells, or between adjacent bacterial cells.A. brasilense Cd were found in root cortical intercellular spaces, but were not detected in either the endodermal layer or in the vascular system. This study proposes that in addition to root surface colonization,A. brasilense Cd forms intercellular associations within wheat roots.     

白芷细胞外钙调素结合蛋白（ECBP21）免疫组化及金标定位分析

ECBP21是我们从白芷悬浮培养细胞外纯化及cDNA克隆的一种钙调素结合蛋白(CaMBP),亦是植物中首次报道的细胞外CaMBP。本实验以大肠杆菌表达的重组ECBP21蛋白为抗原,制备了高效价特异性抗体;随后,利用免疫组化及金标定位技术,研究了ECBP21蛋白组织特异性分布及亚细胞定位。免疫组化结果表明：ECBP21在白芷各组织中均有分布,但在叶、花、花序轴中较多,而在根中较少,并且ECBP21在细胞中多分布于细胞壁区域;免疫胶体金电镜定位结果显示：在白芷花序轴细胞中金颗粒主要分布在细胞壁,表明ECBP21蛋白主要定位于细胞壁区域,从而首次为细胞外CaMBP(ECBP21)的胞外存在提供了直观证据,并为进一步研究其在植物生长发育中的功能提供了初步信息。     

Reprint of "On the feasibility of visualizing ultrasmall gold labels in biological specimens by STEM tomography" [J. Struct. Biol. 159 (2007) 507-522

Labeling with heavy atom clusters attached to antibody fragments is an attractive technique for determining the 3D distribution of specific proteins in cells using electron tomography. However, the small size of the labels makes them very difficult to detect by conventional bright-field electron tomography. Here, we evaluate quantitative scanning transmission electron microscopy (STEM) at a beam voltage of 300 kV for detecting 11-gold atom clusters (Undecagold) and 1.4 nm-diameter nanoparticles (Nanogold) for a variety of specimens and imaging conditions. STEM images as well as tomographic tilt series are simulated by means of the NIST Elastic-Scattering Cross-Section Database for gold clusters embedded in carbon. The simulations indicate that the visibility in 2D of Undecagold clusters in a homogeneous matrix is maximized for low inner collection semi-angles of the STEM annular dark-field detector (15–20 mrad). Furthermore, our calculations show that the visibility of Undecagold in 3D reconstructions is significantly higher than in 2D images for an inhomogeneous matrix corresponding to fluctuations in local density. The measurements demonstrate that it is possible to detect Nanogold particles in plastic sections of tissue freeze-substituted in the presence of osmium. STEM tomography has the potential to localize specific proteins in permeabilized cells using antibody fragments tagged with small heavy atom clusters. Our quantitative analysis provides a framework for determining the detection limits and optimal experimental conditions for localizing these small clusters.     

Oviductal epithelium of the baboon: hormonal control and the immuno-gold localization of oviduct-specific glycoproteins

Oviducts were obtained from a series of cycling and ovariectomized steroid-treated baboons. The lining epithelium of the ampulla and isthmus was analyzed by light and electron microscopy. Both morphological and cytomorphometric analyses revealed that the morphological and functional state of the oviductal epithelium in the baboon is controlled by the ovarian steroids. Additionally, a clear cephalocaudal steroid-responsive gradient was observed when the data from the ampulla and isthmus of the same animal were compared. Within the ampulla, estradiol induced hypertrophy, hyperplasia, ciliogenesis, and secretory activity, whereas adding progesterone to the treatment regimen (+/- estradiol) resulted in atrophy, deciliation, apoptosis, and loss of the secretory activity. These cyclic processes were less evident in the isthmus. We also used an indirect electron microscopic immunogold technique and a previously characterized polyclonal antibody to determine the localization of oviduct-specific glycoproteins. These glycoproteins were present in every secretory granule observed, regardless of oviduct region, electron density, or size of the secretory granule. In summary, our data show that 1) estradiol induces and maintains the mature epithelium of the baboon oviduct, 2) steroid withdrawal or the administration of progesterone causes regression of the epithelium, and 3) the previously identified estradiol-dependent oviduct-specific glycoproteins are synthesized within and released from the secretory epithelial cells.     

Precise localization of an overproduced periplasmic protein in Escherichia coli: use of double immuno-gold labelling

The subcellular localization of beta-lactamase produced by a secretion-cloning vector pIN-III was studied by immunolabelling of frozen thin sections of Escherichia coli. Using double immuno-gold detections and internal reference proteins, it is shown here that beta-lactamase encoded by this vector can be exported and that its overproduction leads to aggregation within the periplasm. This aggregation induces the appearance of electron-dense areas immunolabelled by the antiserum directed against the beta-lactamase at the external side of the cytoplasmic membrane. The overproduced enzyme is also secreted to the medium in vesicles budding from the outer membrane of lpp strains.     

Three-dimensional organization of Rift Valley fever virus revealed by cryoelectron tomography

Rift Valley fever virus (RVFV) is a member of the Bunyaviridae virus family (genus Phlebovirus) and is considered to be one of the most important pathogens in Africa, causing viral zoonoses in livestock and humans. Here, we report the characterization of the three-dimensional structural organization of RVFV vaccine strain MP-12 by cryoelectron tomography. Vitrified-hydrated virions were found to be spherical, with an average diameter of 100 nm. The virus glycoproteins formed cylindrical hollow spikes that clustered into distinct capsomeres. In contrast to previous assertions that RVFV is pleomorphic, the structure of RVFV MP-12 was found to be highly ordered. The three-dimensional map was resolved to a resolution of 6.1 nm, and capsomeres were observed to be arranged on the virus surface in an icosahedral lattice with clear T=12 quasisymmetry. All icosahedral symmetry axes were visible in self-rotation functions calculated using the Fourier transform of the RVFV MP-12 tomogram. To the best of our knowledge, a triangulation number of 12 had previously been reported only for Uukuniemi virus, a bunyavirus also within the Phlebovirus genus. The results presented in this study demonstrate that RVFV MP-12 possesses T=12 icosahedral symmetry and suggest that other members of the Phlebovirus genus, as well as of the Bunyaviridae family, may adopt icosahedral symmetry. Knowledge of the virus architecture may provide a structural template to develop vaccines and diagnostics, since no effective anti-RVFV treatments are available for human use.     

Three-dimensional organization of higher-plant chloroplast thylakoid membranes revealed by electron tomography

The light-harvesting and energy-transducing functions of the chloroplast are performed within an intricate lamellar system of membranes, called thylakoid membranes, which are differentiated into granum and stroma lamellar domains. Using dual-axis electron microscope tomography, we determined the three-dimensional organization of the chloroplast thylakoid membranes within cryo-immobilized, freeze-substituted lettuce (Lactuca sativa) leaves. We found that the grana are built of repeating units that consist of paired layers formed by bifurcations of stroma lamellar sheets, which fuse within the granum body. These units are rotated relative to each other around the axis of the granum cylinder. One of the layers that makes up the pair bends upwards at its edge and fuses with the layer above it, whereas the other layer bends in the opposite direction and merges with the layer below. As a result, each unit in the granum is directly connected to its neighbors as well as to the surrounding stroma lamellae. This highly connected morphology has important consequences for the formation and function of the thylakoid membranes as well as for their stacking/unstacking response to variations in light conditions.     

Three-dimensional architecture of murine rod outer segments determined by cryoelectron tomography

The rod outer segment (ROS) of photoreceptor cells houses all components necessary for phototransduction, a set of biochemical reactions that amplify and propagate a light signal. Theoretical approaches to quantify this process require precise information about the physical boundaries of the ROS. Dimensions of internal structures within the ROS of mammalian species have yet to be determined with the precision required for quantitative considerations. Cryoelectron tomography was utilized to obtain reliable three-dimensional morphological information about this important structure from murine retina. Vitrification of samples permitted imaging of the ROS in a minimally perturbed manner and the preservation of substructures. Tomograms revealed the characteristic highly organized arrangement of disc membranes stacked on top of one another with a surrounding plasma membrane. Distances among the various membrane components of the ROS were measured to define the space available for phototransduction to occur. Reconstruction of segments of the ROS from single-axis tilt series images provided a glimpse into the three-dimensional architecture of this highly differentiated neuron. The reconstructions revealed spacers that likely maintain the proper distance between adjacent discs and between discs and the plasma membrane. Spacers were found distributed throughout the discs, including regions that are distant from the rim region of discs.     

Gold nanomaterials and their potential use as cryo-electron tomography labels

Rapid advances in cryo-electron tomography (cryo-ET) are driving a revolution in cellular structural biology. However, unambiguous identification of specific biomolecules within cellular tomograms remains challenging. Overcoming this obstacle and reliably identifying targets in the crowded cellular environment is of major importance for the understanding of cellular function and is a pre-requisite for high-resolution structural analysis. The use of highly-specific, readily visualised and adjustable labels would help mitigate this issue, improving both data quality and sample throughput. While progress has been made in cryo-CLEM and in the development of cloneable high-density tags, technical issues persist and a robust ‘cryo-GFP’ remains elusive. Readily-synthesized gold nanomaterials conjugated to small ‘affinity modules’ may represent a solution. The synthesis of materials including gold nanoclusters (AuNCs) and gold nanoparticles (AuNPs) is increasingly well understood and is now within the capabilities of non-specialist laboratories. The remarkable chemical and photophysical properties of <3nm diameter nanomaterials and their emergence as tools with widespread biomedical application presents significant opportunities to the cryo-microscopy community. In this review, we will outline developments in the synthesis, functionalisation and labelling uses of both AuNPs and AuNCs in cryo-ET, while discussing their potential as multi-modal probes for cryo-CLEM.     

Three-dimensional computed tomography of the mummy Wenuhotep

Computed tomography allows cross-sectional imaging of anthropological as well as clinical subjects. Recently, technical innovations have made three-dimensional reconstruction of these images feasible. We performed two-dimensional and three-dimensional computed tomography of a Late Period Egyptian mummy to reexamine findings seen on previous radiographic studies and to evaluate the usefulness of these techniques in paleopathology. Two-dimensional images provided excellent anatomic detail. There was graphic depiction of the mummification process that corroborated information previously obtained from Egyptological studies. Three-dimensional reconstruction provided images of facial features as if the mummy had been unwrapped. Three-dimensional computed tomography is a useful method of nondestructively evaluating paleopathological remains, and it may yield information not obtainable by any other means.     

Three-dimensional structures of pathogenic and saprophytic Leptospira species revealed by cryo-electron tomography

Leptospira interrogans is the primary causative agent of the most widespread zoonotic disease, leptospirosis. An in-depth structural characterization of L. interrogans is needed to understand its biology and pathogenesis. In this study, cryo-electron tomography (cryo-ET) was used to compare pathogenic and saprophytic species and examine the unique morphological features of this group of bacteria. Specifically, our study revealed a structural difference between the cell envelopes of L. interrogans and Leptospira biflexa involving variations in the lipopolysaccharide (LPS) layer. Through cryo-ET and subvolume averaging, we determined the first three-dimensional (3-D) structure of the flagellar motor of leptospira, with novel features in the flagellar C ring, export apparatus, and stator. Together with direct visualization of chemoreceptor arrays, DNA packing, periplasmic filaments, spherical cytoplasmic bodies, and a unique "cap" at the cell end, this report provides structural insights into these fascinating Leptospira species.     

Three-dimensional reconstruction of axonemal outer dynein arms in situ by electron tomography

We present here for the first time a 3D reconstruction of in situ axonemal outer dynein arms. This reconstruction has been obtained by electron tomography applied to a series of tilted images collected from metal replicas of rapidly frozen, cryofractured, and metal-replicated sperm axonemes of the cecidomid dipteran Monarthropalpus flavus. This peculiar axonemal model consists of several microtubular laminae that proved to be particularly suitable for this type of analysis. These laminae are sufficiently planar to allow the visualization of many dynein molecules within the same fracture face, allowing us to recover a significant number of equivalent objects and to improve the signal-to-noise ratio of the reconstruction by applying advanced averaging protocols. The 3D model we obtained showed the following interesting structural features: First, each dynein arm has two head domains that are almost parallel and are obliquely oriented with respect to the longitudinal axis of microtubules. The two heads are therefore positioned at different distances from the surface of the A-tubule. Second, each head domain consists of a series of globular subdomains that are positioned on the same plane. Third, a stalk domain originates as a conical region from the proximal head and ends with a small globular domain that contacts the B-tubule. Fourth, the stem region comprises several globular subdomains and presents two distinct points of anchorage to the surface of the A-tubule. Finally, and most importantly, contrary to what has been observed in isolated dynein molecules adsorbed to flat surfaces, the stalk and the stem domains are not in the same plane as the head.     

Three-dimensional elemental mapping of phosphorus by quantitative electron spectroscopic tomography (QuEST)

We describe the development of quantitative electron spectroscopic tomography (QuEST), which provides 3-D distributions of elements on a nanometer scale. Specifically, it is shown that QuEST can be applied to map the distribution of phosphorus in unstained sections of embedded cells. A series of 2-D elemental maps is derived from images recorded in the energy filtering transmission electron microscope for a range of specimen tilt angles. A quantitative 3-D elemental distribution is then reconstructed from the elemental tilt series. To obtain accurate quantitative elemental distributions it is necessary to correct for plural inelastic scattering at the phosphorus L(2,3) edge, which is achieved by acquiring unfiltered and zero-loss images at each tilt angle. The data are acquired automatically using a cross correlation technique to correct for specimen drift and focus change between successive tilt angles. An algorithm based on the simultaneous iterative reconstruction technique (SIRT) is implemented to obtain quantitative information about the number of phosphorus atoms associated with each voxel in the reconstructed volume. We assess the accuracy of QuEST by determining the phosphorus content of ribosomes in a eukaryotic cell, and then apply it to estimate the density of nucleic acid in chromatin of the cell's nucleus. From our experimental data, we estimate that the sensitivity for detecting phosphorus is 20 atoms in a 2.7 nm-sized voxel.     

Correlation between immuno-gold labels and activities of the cytochrome-c oxidase (aa3-type) in membranes of salt stressed cyanobactria

Abstract Anacystis nidulans ( Synechococcus PCC6301) and Synechocystis PCC6803 were grown photoautotrophically in a turbido-statically operated chemostat at a constant cell concentration of 2.0±0.3 μ l packed cell mass per ml in the presence of elevated NaCl concentrations up to 0.5 M ('salt stress'). The impact of salt stress on ccytochrome- c oxidase (EC 1.9.3.1) was` studied on isolated and purified membranes, and by immuno-gold labeling of thin-sectioned whole cells ATPase activities of membranes isolated and separated from cells under varying salt stress were also measured. Anacystis and Synechocystis adapted to the presence of 0.5 M NaCl in the medium with lag phases of 2 days and 2 hours, respectively. Both isolated plasma and thylakoid membranes from salt adapted Synechocystis displayed 5- to 8-times enhanced cytcytochrome- c oxidase activities while in Anacystis the effect was restricted to the plasma membrane. In either case less than proportionately increased counts of immuno-gold labeled cytochrome- c oxidase molecules in the respective membranes were obtained, the additional increment being attributed to the increased lipid content of the membranes from salt-adapted cells, leading to increased specific activities of the enzyme compared to control cells. ATPase activity of plasma membranes from Synechocystis was far more increased than of those from Anacystis while in thylakoid membranes the differentiating effect was less pronounced. Our results are discussed in terms of distinct strategies for salt adaptation in the two cyanobacterial species whereby in Anacystis the plasma membrane-bound respiratory chain and in Synechocystis the plasma membrane-bound ATPase(s) play the major role for plasma membrane energization which, in turn, is necessary for the active exclusion of sodium from the cell interior.     

Improved visualization of the immunoreactive hypothalamo-neurohypophysial system by use of immuno-gold techniques

Summary Ultrastructural post-embedding immuno-gold techniques were applied to the supraoptic nucleus and the neurohypophysis of mice and rats. The primary antibodies were three different monoclonal antineurophysins, used in protein A-gold and immunoglobulin-gold procedures. Conventional plastic embedding as well as hydrophilic media (L.R. White) were used; non-osmicated and osmicated tissues were immunolabeled; sodium metaperiodate oxidation was used, but was not essential for immunolabeling.Vasopressinergic and oxytocinergic NSGs were identified by the specific immunoreactivity of their respective neurophysins on adjacent thin sections, and by sequential double labeling on the same thin section using two different antibodies associated with gold probes of different diameters. The immunoidentification indicates that vasopressin NSGs can additionally be differentiated as larger, with more electron-dense matrix, and susceptible to damage by sodium metaperiodate.The only organelles consistently labeled were neurosecretory granules (NSGs), either intact or within lysosomal configurations. Some lysosomal dense bodies were immunoreactive even when discrete NSGs were no longer morphologically recognisable within them. Labeled NSGs were located within neuronal cell bodies, along axonal shafts and within axonal swellings and endings; occasionally immunoreactive NSGs were observed within synaptic boutons. Labeling intensity was semi-quantitatively gauged by counting gold particles in relation to numbers of NSGs per axonal varicosity.The precise localisation achieved with particulate immunogold labeling surpasses that previously obtained with diffuse electron-dense immunoreaction products.     

Three-dimensional imaging of the highly bent architecture of Bdellovibrio bacteriovorus by using cryo-electron tomography

Bdellovibrio bacteriovorus cells are small deltaproteobacterial cells that feed on other gram-negative bacteria, including human pathogens. Using cryo-electron tomography, we demonstrated that B. bacteriovorus cells are capable of substantial flexibility and local deformation of the outer and inner membranes without loss of cell integrity. These shape changes can occur in less than 2 min, and analysis of the internal architecture of highly bent cells showed that the overall distribution of molecular machines and the nucleoid is similar to that in moderately bent cells. B. bacteriovorus cells appear to contain an extensive internal network of short and long filamentous structures. We propose that rearrangements of these structures, in combination with the unique properties of the cell envelope, may underlie the remarkable ability of B. bacteriovorus cells to find and enter bacterial prey.     

Three-dimensional gas exchange pathways in pome fruit characterized by synchrotron x-ray computed tomography

Our understanding of the gas exchange mechanisms in plant organs critically depends on insights in the three-dimensional (3-D) structural arrangement of cells and voids. Using synchrotron radiation x-ray tomography, we obtained for the first time high-contrast 3-D absorption images of in vivo fruit tissues of high moisture content at 1.4-microm resolution and 3-D phase contrast images of cell assemblies at a resolution as low as 0.7 microm, enabling visualization of individual cell morphology, cell walls, and entire void networks that were previously unknown. Intercellular spaces were always clear of water. The apple (Malus domestica) cortex contains considerably larger parenchyma cells and voids than pear (Pyrus communis) parenchyma. Voids in apple often are larger than the surrounding cells and some cells are not connected to void spaces. The main voids in apple stretch hundreds of micrometers but are disconnected. Voids in pear cortex tissue are always smaller than parenchyma cells, but each cell is surrounded by a tight and continuous network of voids, except near brachyssclereid groups. Vascular and dermal tissues were also measured. The visualized network architecture was consistent over different picking dates and shelf life. The differences in void fraction (5.1% for pear cortex and 23.0% for apple cortex) and in gas network architecture helps explain the ability of tissues to facilitate or impede gas exchange. Structural changes and anisotropy of tissues may eventually lead to physiological disorders. A combined tomography and internal gas analysis during growth are needed to make progress on the understanding of void formation in fruit.     

Three-dimensional cranial surface reconstructions using high-resolution computed tomography

Until recently, there has been no satisfactory way for anthropologists to visualize intracranial morphology in more than two dimensions without actually "invading" the skull in some manner. Images provided by conventional x-ray and computed tomography (CT) scans are often abstract, flat, two-dimensional representations that fail to reveal three-dimensional relationships. We describe new computer-imaging techniques that reconstruct three-dimensional images from sequential series of narrowly collimated (1-2 mm), high-resolution CT scans of the skull. These computed images represent three-dimensional surface data and can be viewed from any direction. Depth information is encoded in gray scale. In addition, selected portions of the anatomy can be "removed", i.e., made transparent, to allow visualization of previously hidden intracranial morphology. Since the geometric data obtained with the CT scanner are precise, parameters such as linear distances, angles, areas, and volumes can be accurately (and instantaneously) generated. The power and versatility of these computer-imaging techniques are demonstrated by examining living subjects with major craniofacial dysmorphology (Treacher-Collins syndrome and unilateral coronal synostosis); an anthropoid osteological specimen (Gorilla); and a fossil mammal skull.