Row-like organization of ATP synthase in intact mitochondria determined by cryo-electron tomography

The fine structure of intact, close-to-spherical mitochondria from the alga Polytomella was visualized by dual-axis cryo-electron tomography. The supramolecular organization of dimeric ATP synthase in the cristae membranes was investigated by averaging subvolumes of tomograms and 3D details at ∼ 6 nm resolution were revealed. Oligomeric ATP synthase is composed of rows of dimers at 12 nm intervals; the dimers make a slight angle along the row. In addition, the main features of monomeric ATP synthase, such as the conically shaped F₁ headpiece, central stalk and stator were revealed. This demonstrates the capability of dual-axis electron tomography to unravel details of proteins and their interactions in complete organelles.     

生物三维电子显微学进入全新高速发展时期

生物三维电子显微学主要由三个部分组成——电子晶体学、单颗粒技术和电子断层成像术,其结构解析对象的尺度范围介于x射线晶体学与光学显微镜之间,适合从蛋白质分子结构到细胞和组织结构的解析。以冷冻电镜技术与三维重构技术为基础的低温电子显微学代表了生物电子显微学的前沿。低温单颗粒技术对于高度对称的病毒颗粒的解析最近已达到3．8A分辨率,正在成为解析分子量很大的蛋白质复合体高分辨结构的有效技术手段。低温电子断层成像技术目前对于真核细胞样品的结构解析已达到约40A的分辨率,在今后5年有望达到20A。这样,把x射线晶体学、NMR以及电镜三维重构获得的蛋白质分子及复合体的高分辨率的结构,锚定到较低分辨率的电子断层成像图像中,从而在细胞水平上获得高精确的蛋白质空间定位和原子分辨率的蛋白质相互作用的结构信息。这将成为把分子水平的结构研究与细胞水平的生命活动衔接起来的可行途径。     

The Internal Architecture of Leukocyte Lipid Body Organelles Captured by Three-Dimensional Electron Microscopy Tomography

Lipid bodies (LBs), also known as lipid droplets, are complex organelles of all eukaryotic cells linked to a variety of biological functions as well as to the development of human diseases. In cells from the immune system, such as eosinophils, neutrophils and macrophages, LBs are rapidly formed in the cytoplasm in response to inflammatory and infectious diseases and are sites of synthesis of eicosanoid lipid mediators. However, little is known about the structural organization of these organelles. It is unclear whether leukocyte LBs contain a hydrophobic core of neutral lipids as found in lipid droplets from adipocytes and how diverse proteins, including enzymes involved in eicosanoid formation, incorporate into LBs. Here, leukocyte LB ultrastructure was studied in detail by conventional transmission electron microscopy (TEM), immunogold EM and electron tomography. By careful analysis of the two-dimensional ultrastructure of LBs from human blood eosinophils under different conditions, we identified membranous structures within LBs in both resting and activated cells. Cyclooxygenase, a membrane inserted protein that catalyzes the first step in prostaglandin synthesis, was localized throughout the internum of LBs. We used fully automated dual-axis electron tomography to study the three-dimensional architecture of LBs in high resolution. By tracking 4 nm-thick serial digital sections we found that leukocyte LBs enclose an intricate system of membranes within their “cores”. After computational reconstruction, we showed that these membranes are organized as a network of tubules which resemble the endoplasmic reticulum (ER). Our findings explain how membrane-bound proteins interact and are spatially arranged within LB “cores” and support a model for LB formation by incorporating cytoplasmic membranes of the ER, instead of the conventional view that LBs emerge from the ER leaflets. This is important to understand the functional capabilities of leukocyte LBs in health and during diverse diseases in which these organelles are functionally involved.     

Use of cryo-negative staining in tomographic reconstruction of biological objects: application to T4 bacteriophage

Recent advances in electron microscopy and image analysis techniques have resulted in the development of tomography, which makes possible the study of structures neither accessible to X-ray crystallography nor nuclear magnetic resonance. However, the use of tomography to study biological structures, ranging from 100 to 500 nm, requires developments in sample preparation and image analysis. Indeed, cryo-electron tomography present two major drawbacks: the low contrast of recorded images and the sample radiation damage. In the present work we have tested, on T4 bacteriophage samples, the use of a new preparation technique, cryo-negative staining, which reduces the radiation damage while preserving a high signal-to-noise ratio. Our results demonstrate that the combination of cryo-negative staining in tomography with standard cryo-microscopy and single particle analysis results in a methodological approach that could be useful in the study of biological structures ranging in the T4 bacteriophage size.     

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.     

Single versus dual-axis cryo-electron tomography of microtubules assembled in vitro: Limits and perspectives

Single-axis cryo-electron tomography of vitrified specimens has become a method of choice to reconstruct in three dimensions macromolecular assemblies in their cellular context or prepared from purified components. Here, we asked how a dual-axis acquisition scheme would improve three-dimensional reconstructions of microtubules assembled in vitro. We show that in single-axis tomograms, microtubules oriented close to the perpendicular of the tilt axis display diminished contrast, and ultimately transform into sets of parallel lines oriented in the direction of the electron beam when observed in cross-section. Analysis of their three-dimensional Fourier transform indicates that this imaging artifact is due to a decrease in the angular sampling of their equatorial components. Although the second orthogonal series does not fully complement the first one at the specimen level due to increased radiation damage, it still allows elongated features oriented in any directions to be correctly reconstructed, which might be essential for highly heterogeneous specimens such as cells.     

Stereology meets electron tomography: towards quantitative 3D electron microscopy

Stereological tools are the gold standard for accurate (i.e., unbiased) and precise quantification of any microscopic sample. The past decades have provided a broad spectrum of tools to estimate a variety of parameters such as volumes, surfaces, lengths, and numbers. Some of them require pairs of parallel sections that can be produced by either physical or optical sectioning, with optical sectioning being much more efficient when applicable. Unfortunately, transmission electron microscopy could not fully profit from these riches, mainly because of the large depth of field. Hence, optical sectioning was a long-time desire for electron microscopists. This desire was fulfilled with the development of electron tomography that yield stacks of slices from electron microscopic sections. Now, parallel optical slices of a previously unimagined small thickness (2-5 nm axial resolution) can be produced. These optical slices minimize problems related to overprojection effects, and allow for direct stereological analysis, e.g., volume estimation with the Cavalieri principle and number estimation with the optical disector method. Here, we demonstrate that the symbiosis of stereology and electron tomography is an easy and efficient way for quantitative analysis at the electron microscopic level. We call this approach quantitative 3D electron microscopy.     

A novel dual-axis iterative algorithm for electron tomography

A new algorithm for computing electron microscopy tomograms which combines iterative methods with dual-axis geometry is presented. Initial modelling using test data shows several improvements over both the weighted back-projection and simultaneous iterative reconstruction technique methods, with increased stability and tomogram fidelity under high-noise conditions. Preliminary experimental dual-axis reconstructions confirm the viability of the new algorithm.     

Reprint of “Stereology meets electron tomography: towards quantitative 3D electron microscopy” [J. Struct. Biol. 159 (2007) 443–450]

Stereological tools are the gold standard for accurate (i.e., unbiased) and precise quantification of any microscopic sample. The past decades have provided a broad spectrum of tools to estimate a variety of parameters such as volumes, surfaces, lengths, and numbers. Some of them require pairs of parallel sections that can be produced by either physical or optical sectioning, with optical sectioning being much more efficient when applicable. Unfortunately, transmission electron microscopy could not fully profit from these riches, mainly because of the large depth of field. Hence, optical sectioning was a long-time desire for electron microscopists.This desire was fulfilled with the development of electron tomography that yield stacks of slices from electron microscopic sections. Now, parallel optical slices of a previously unimagined small thickness (2–5 nm axial resolution) can be produced. These optical slices minimize problems related to overprojection effects, and allow for direct stereological analysis, e.g., volume estimation with the Cavalieri principle and number estimation with the optical disector method.Here, we demonstrate that the symbiosis of stereology and electron tomography is an easy and efficient way for quantitative analysis at the electron microscopic level. We call this approach quantitative 3D electron microscopy.     

Classification and 3D averaging with missing wedge correction in biological electron tomography

Strategies for the determination of 3D structures of biological macromolecules using electron crystallography and single-particle electron microscopy utilize powerful tools for the averaging of information obtained from 2D projection images of structurally homogeneous specimens. In contrast, electron tomographic approaches have often been used to study the 3D structures of heterogeneous, one-of-a-kind objects such as whole cells where image-averaging strategies are not applicable. Complex entities such as cells and viruses, nevertheless, contain multiple copies of numerous macromolecules that can individually be subjected to 3D averaging. Here we present a complete framework for alignment, classification, and averaging of volumes derived by electron tomography that is computationally efficient and effectively accounts for the missing wedge that is inherent to limited-angle electron tomography. Modeling the missing data as a multiplying mask in reciprocal space we show that the effect of the missing wedge can be accounted for seamlessly in all alignment and classification operations. We solve the alignment problem using the convolution theorem in harmonic analysis, thus eliminating the need for approaches that require exhaustive angular search, and adopt an iterative approach to alignment and classification that does not require the use of external references. We demonstrate that our method can be successfully applied for 3D classification and averaging of phantom volumes as well as experimentally obtained tomograms of GroEL where the outcomes of the analysis can be quantitatively compared against the expected results.     

Effects of liquid stimuli on dual-axis swallowing accelerometry signals in a healthy population

Background  

Dual-axis swallowing accelerometry has recently been proposed as a tool for non-invasive analysis of swallowing function. Although swallowing is known to be physiologically modifiable by the type of food or liquid (i.e., stimuli), the effects of stimuli on dual-axis accelerometry signals have never been thoroughly investigated. Thus, the objective of this study was to investigate stimulus effects on dual-axis accelerometry signal characteristics. Signals were acquired from 17 healthy participants while swallowing 4 different stimuli: water, nectar-thick and honey-thick apple juices, and a thin-liquid barium suspension. Two swallowing tasks were examined: discrete and sequential. A variety of features were extracted in the time and time-frequency domains after swallow segmentation and pre-processing. A separate Friedman test was conducted for each feature and for each swallowing task.     

A novel approach for intracellular 3D immuno-labeling for electron tomography

Electron tomography (ET) is an indispensable high-resolution tool for three dimensional (3D) imaging in cell biology. When applied to immuno-labeled cells, ET can provide essential insights in both the cellular architecture and the dynamics. Current protocols for 3D immuno-labeling of intracellular antigens include permeabilization steps that cause random, extensive cell membrane disruption. This permeabilization results in a poor cell ultrastructure, limiting the usefulness of the specimens for high-resolution studies. Here we describe a novel method, based on a well-controlled permeabilization by targeted laser cell perforation, that allows for the 3D immuno-localization of cytoplasmic antigens in cultured cells. The approach is unique since it is applicable to both chemically and cryo-fixed cells and leads to a superior ultrastructural preservation for electron microscopy and tomography.     

Three-dimensional structure of the Trypanosome flagellum suggests that the paraflagellar rod functions as a biomechanical spring

Flagellum motility is critical for normal human development and for transmission of pathogenic protozoa that cause tremendous human suffering worldwide. Biophysical principles underlying motility of eukaryotic flagella are conserved from protists to vertebrates. However, individual cells exhibit diverse waveforms that depend on cell-specific elaborations on basic flagellum architecture. Trypanosoma brucei is a uniflagellated protozoan parasite that causes African sleeping sickness. The T. brucei flagellum is comprised of a 9+2 axoneme and an extra-axonemal paraflagellar rod (PFR), but the three-dimensional (3D) arrangement of the underlying structural units is poorly defined. Here, we use dual-axis electron tomography to determine the 3D architecture of the T. brucei flagellum. We define the T. brucei axonemal repeating unit. We observe direct connections between the PFR and axonemal dyneins, suggesting a mechanism by which mechanochemical signals may be transmitted from the PFR to axonemal dyneins. We find that the PFR itself is comprised of overlapping laths organized into distinct zones that are connected through twisting elements at the zonal interfaces. The overall structure has an underlying 57 nm repeating unit. Biomechanical properties inferred from PFR structure lead us to propose that the PFR functions as a biomechanical spring that may store and transmit energy derived from axonemal beating. These findings provide insight into the structural foundations that underlie the distinctive flagellar waveform that is a hallmark of T. brucei cell motility.     

108 Individual-particle electron tomography: a method for studying macromolecule dynamics

In solution, macromolecules are naturally flexible and dynamic. Dynamic personalities and structural heterogeneities of macromolecules are essential to understanding their proper function (Karplus & Kuriyan, 2005). However, structural determination of dynamic/heterogeneous macromolecules is limited by current technology such as: X-ray crystallography, nuclear magnetic resonance spectrum, small angle scattering, and electron microscopy single-particle reconstruction. A common weakness of all current techniques is requiring an averaged signal from thousands to millions of different macromolecules. Using averaged “signal” must involve in an assumption that macromolecules remain in identical structures or few identical conformations. This assumption is a good estimate for some macromolecule that have a rigid body, but not for most macromolecules that have “soft”, flexible, and dynamic body, such as lipoproteins and antibodies. An ideal approach for structure determination regardless of macromolecular dynamics is to use non-averaged signal, i.e. the signal from a single macromolecule itself. We developed a ‘‘focused electron tomography reconstruction’’ (FETR) algorithm to improve the resolution by decreasing the reconstructing image size so that it contains only a single-instance macromolecule (Zhang & Ren, 2012). FETR can tolerate certain levels of image distortion and measuring tilt-errors, and can also precisely determine the translational parameters via an iterative refinement process that contains a series of automatically generated dynamic filters and masks. Since this approach can obtain the structure of a single-instance macromolecule, we named it individual-particle electron tomography (IPET) as a new robust strategy/approach that does not require a pre-given initial model, class averaging of multiple molecules or an extended ordered lattice, but can tolerate small tilt-errors for high-resolution single ‘‘snapshot’’ of molecule structure determination (Zhang & Ren, 2012). FETR/IPET provides a completely new opportunity for a single-macromolecule structure determination, and could be used to study the dynamic character, equilibrium fluctuation, to reveal macromolecular mechanism, and even to track the intermediate state of the reaction of macromolecules (Zhang et al., 2010; Zhang & Ren, 2010).     

Three‐Phase Multiscale Modeling of a LiCoO2 Cathode: Combining the Advantages of FIB–SEM Imaging and X‐Ray Tomography

LiCoO₂ electrodes contain three phases, or domains, each having specific‐intended functions: ion‐conducting pore space, lithium‐ion‐reacting active material, and electron conducting carbon‐binder domain (CBD). Transport processes take place in all domains on different characteristic length scales: from the micrometer scale in the active material grains through to the nanopores in the carbon‐binder phase. Consequently, more than one imaging approach must be utilized to obtain a hierarchical geometric representation of the electrode. An approach incorporating information from the micro‐ and nanoscale to calculate 3D transport‐relevant properties in a large‐reconstructed active domain is presented. Advantages of focused ion beam/scanning electron microscopy imaging and X‐ray tomography combined by a spatial stochastic model, validated with an artificially produced reference structure are used. This novel approach leads to significantly different transport relevant properties compared with previous tomographic approaches: nanoporosity of the CBD leads to up to 42% additional contact area between active material and pore space and increases ionic conduction by a factor of up to 3.6. The results show that nanoporosity within the CBD cannot be neglected.     

The possible use of new technologies for treating some diseases at the atomic-molecular level

Transitions from the normal to the pathological state can be studied at the atomic-molecular level simultaneously with treatment with electron spin resonance electrodialysis and nuclear magnetic resonance electrodialysis under the conditions of biological specimen cultivation, with simultaneous recording of microcalorimetry parameters. It is reasonable to test the possibility of application of some diagnostic recording devices as therapeutic agents. Modeling of the conditions of x-ray tomography permits these devices to also be used to determine the safety of x-ray tomography at the atomic-molecular level. This work shows the possibility of studying early rearrangements and states at the premorbid stage.     

IPET and FETR: experimental approach for studying molecular structure dynamics by cryo-electron tomography of a single-molecule structure

The dynamic personalities and structural heterogeneity of proteins are essential for proper functioning. Structural determination of dynamic/heterogeneous proteins is limited by conventional approaches of X-ray and electron microscopy (EM) of single-particle reconstruction that require an average from thousands to millions different molecules. Cryo-electron tomography (cryoET) is an approach to determine three-dimensional (3D) reconstruction of a single and unique biological object such as bacteria and cells, by imaging the object from a series of tilting angles. However, cconventional reconstruction methods use large-size whole-micrographs that are limited by reconstruction resolution (lower than 20 Å), especially for small and low-symmetric molecule (<400 kDa). In this study, we demonstrated the adverse effects from image distortion and the measuring tilt-errors (including tilt-axis and tilt-angle errors) both play a major role in limiting the reconstruction resolution. Therefore, we developed a “focused electron tomography reconstruction” (FETR) algorithm to improve the resolution by decreasing the reconstructing image size so that it contains only a single-instance protein. FETR can tolerate certain levels of image-distortion and measuring tilt-errors, and can also precisely determine the translational parameters via an iterative refinement process that contains a series of automatically generated dynamic filters and masks. To describe this method, a set of simulated cryoET images was employed; to validate this approach, the real experimental images from negative-staining and cryoET were used. Since this approach can obtain the structure of a single-instance molecule/particle, we named it individual-particle electron tomography (IPET) as a new robust strategy/approach that does not require a pre-given initial model, class averaging of multiple molecules or an extended ordered lattice, but can tolerate small tilt-errors for high-resolution single “snapshot” molecule structure determination. Thus, FETR/IPET provides a completely new opportunity for a single-molecule structure determination, and could be used to study the dynamic character and equilibrium fluctuation of macromolecules.     

Three-dimensional imaging of biological complexity

Over the past 5 years, thanks to advances in both instrumentation and computational speed, three-dimensional imaging techniques using the electron microscope have been greatly improved in two areas: electron tomography of cell organelles or cell sections and reconstruction of macromolecules from single particles. Ice embedment has brought a breakthrough in the degree of preservation of specimens under close-to-native conditions. The current challenge is to push the resolution of electron tomographic imaging to a point where macromolecular signatures can be recognized within the cellular context. We show first progress toward this goal by examples in two areas of application: the structure of the muscle triad junction and the architecture and fine structure of mitochondria. As techniques of cryo-microtomy are perfected, we hope to be able to apply tomography to high-pressure frozen sections of tissue.