Towards neural circuit reconstruction with volume electron microscopy techniques

Electron microscopy is the only currently available technique with a resolution adequate to identify and follow every axon and dendrite in dense neuropil. Reconstructions of large volumes of neural tissue, necessary to reconstruct even local neural circuits, have, however, been inhibited by the daunting task of serially sectioning and reconstructing thousands of sections. Recent technological developments have improved the quality of volume electron microscopy data and automated its acquisition. This opens up the prospect of reconstructing almost complete invertebrate and sizable fractions of vertebrate nervous systems. Such reconstructions of complete neural wiring diagrams could rekindle the tradition of relating neural function to the underlying neuroanatomical circuitry.     

TomoJ: tomography software for three-dimensional reconstruction in transmission electron microscopy

Background  

Transmission electron tomography is an increasingly common three-dimensional electron microscopy approach that can provide new insights into the structure of subcellular components. Transmission electron tomography fills the gap between high resolution structural methods (X-ray diffraction or nuclear magnetic resonance) and optical microscopy. We developed new software for transmission electron tomography, TomoJ. TomoJ is a plug-in for the now standard image analysis and processing software for optical microscopy, ImageJ.     

Fitting of high-resolution structures into electron microscopy reconstruction images

Dynamic macromolecular assemblies, such as ribosomes, viruses, and muscle protein complexes, are often more amenable to visualization by electron microscopy than by high-resolution X-ray crystallography or NMR. When high-resolution structures of component structures are available, it is possible to build an atomic model that gives information about the molecular interactions at greater detail than the experimental resolution, due to constraints of modeling placed upon the interpretation. There are now several competing computational methods to search systematically for orientations and positions of components that match the experimental image density, and continuing developments will be reviewed. Attention is now also moving toward the related task of optimization, with flexible and/or multifragment models and sometimes with stereochemically restrained refinement methods. This paper will review the various approaches and describe advances in the authors' methods and applications of real-space refinement.     

High-contrast en bloc staining of neuronal tissue for field emission scanning electron microscopy

Conventional heavy metal poststaining methods on thin sections lend contrast but often cause contamination. To avoid this problem, we tested several en bloc staining techniques to contrast tissue in serial sections mounted on solid substrates for examination by field emission scanning electron microscopy (FESEM). Because FESEM section imaging requires that specimens have higher contrast and greater electrical conductivity than transmission electron microscopy (TEM) samples, our technique uses osmium impregnation (OTO) to make the samples conductive while heavily staining membranes for segmentation studies. Combining this step with other classic heavy metal en bloc stains, including uranyl acetate (UA), lead aspartate, copper sulfate and lead citrate, produced clean, highly contrasted TEM and scanning electron microscopy (SEM) samples of insect, fish and mammalian nervous systems. This protocol takes 7-15 d to prepare resin-embedded tissue, cut sections and produce serial section images.     

Automated reconstruction of three-dimensional neuronal morphology from laser scanning microscopy images

Experimental and theoretical studies demonstrate that both global dendritic branching topology and fine spine geometry are crucial determinants of neuronal function, its plasticity and pathology. Importantly, simulation studies indicate that the interaction between local and global morphologic properties is pivotal in determining dendritic information processing and the induction of synapse-specific plasticity. The ability to reconstruct and quantify dendritic processes at high resolution is therefore an essential prerequisite to understanding the structural determinants of neuronal function. Existing methods of digitizing 3D neuronal structure use interactive manual computer tracing from 2D microscopy images. This method is time-consuming, subjective and lacks precision. In particular, fine details of dendritic varicosities, continuous dendritic taper, and spine morphology cannot be captured by these systems. We describe a technique for automated reconstruction of 3D neuronal morphology from multiple stacks of tiled confocal and multiphoton laser scanning microscopy (CLSM and MPLSM) images. The system is capable of representing both global and local structural variations, including gross dendritic branching topology, dendritic varicosities, and fine spine morphology with sufficient resolution for accurate 3D morphometric analyses and realistic biophysical compartment modeling. Our system provides a much needed tool for automated digitization and reconstruction of 3D neuronal morphology that reliably captures detail on spatial scales spanning several orders of magnitude, that avoids the subjective errors that arise during manual tracing with existing digitization systems, and that runs on a standard desktop workstation.     

Three-dimensional reconstruction of maltoporin from electron microscopy and image processing.

Two dimensional crystals of maltoporin (or phage lambda receptor) were obtained by reconstitution of purified maltoporin trimers and Escherichia coli phospholipids by detergent dialysis. Two different trimer packing forms were observed. One was hexagonal (a = 7.8 nm) and one rectangular (a = 7.8 nm, b = 13.6 nm). In this paper we describe the three-dimensional structure of maltoporin, deduced from the study of the rectangular form by electron microscopy and image processing. At a resolution of approximately 2.5 nm, maltoporin trimers form aqueous channel triplets which appear to merge into a single outlet at the periplasmic surface of the outer membrane. The pore defined by maltoporin has a similar structure to that outlined by the matrix protein. From the results of functional studies by conductance measurement, it is concluded that the three channels defined by maltoporin act, contrary to those formed by the porin (OmpF protein), as a single conducting unit. A tentative outline of the maltoporin promoter is given. Maltoporin appears to be constituted by three different domains: a major rod-like domain spanning the membrane, a minor domain located near the periplasmic surface of the membrane and finally a central domain responsible for the splitting of the channel.     

Electronic detectors for electron microscopy

Electron microscopy (EM) is an important tool for high-resolution structure determination in applications ranging from condensed matter to biology. Electronic detectors are now used in most applications in EM as they offer convenience and immediate feedback that is not possible with film or image plates. The earliest forms of electronic detector used routinely in transmission electron microscopy (TEM) were charge coupled devices (CCDs) and for many applications these remain perfectly adequate. There are however applications, such as the study of radiation-sensitive biological samples, where film is still used and improved detectors would be of great value. The emphasis in this review is therefore on detectors for use in such applications. Two of the most promising candidates for improved detection are: monolithic active pixel sensors (MAPS) and hybrid pixel detectors (of which Medipix2 was chosen for this study). From the studies described in this review, a back-thinned MAPS detector appears well suited to replace film in for the study of radiation-sensitive samples at 300 keV, while Medipix2 is suited to use at lower energies and especially in situations with very low count rates. The performance of a detector depends on the energy of electrons to be recorded, which in turn is dependent on the application it is being used for; results are described for a wide range of electron energies ranging from 40 to 300 keV. The basic properties of detectors are discussed in terms of their modulation transfer function (MTF) and detective quantum efficiency (DQE) as a function of spatial frequency.     

Electronic detectors for electron microscopy

Due to the increasing popularity of electron cryo-microscopy (cryoEM) in the structural analysis of large biological molecules and macro-molecular complexes and the need for simple, rapid and efficient readout, there is a persuasive need for improved detectors. Commercial detectors, based on phosphor/fibre optics-coupled CCDs, provide adequate performance for many applications, including electron diffraction. However, due to intrinsic light scattering within the phosphor, spatial resolution is limited. Careful measurements suggest that CCDs have superior performance at lower resolution while all agree that film is still superior at higher resolution. Consequently, new detectors are needed based on more direct detection, thus avoiding the intermediate light conversion step required for CCDs. Two types of direct detectors are discussed in this review. First, there are detectors based on hybrid technology employing a separate pixellated sensor and readout electronics connected with bump bonds-hybrid pixel detectors (HPDs). Second, there are detectors, which are monolithic in that sensor and readout are all in one plane (monolithic active pixel sensor, MAPS). Our discussion is centred on the main parameters of interest to cryoEM users, viz. detective quantum efficiency (DQE), resolution or modulation transfer function (MTF), robustness against radiation damage, speed of readout, signal-to-noise ratio (SNR) and the number of independent pixels available for a given detector.     

Isolation, electron microscopy and 3D reconstruction of invertebrate muscle myofilaments

Understanding the molecular mechanism of muscle contraction and its regulation has been greatly influenced and aided by studies of myofilament structure in invertebrate muscles. Invertebrates are easily obtained and cover a broad spectrum of species and functional specializations. The thick (myosin-containing) filaments from some invertebrates are especially stable and simple in structure and thus much more amenable to structural analysis than those of vertebrates. Comparative studies of invertebrate filaments by electron microscopy and image processing have provided important generalizations of muscle molecular structure and function. This article reviews methods for preparing thick and thin filaments from invertebrate muscle, for imaging filaments by electron microscopy, and for determining their three dimensional structure by image processing. It also highlights some of the key insights into filament function that have come from these studies.     

Cortactin binding to F-actin revealed by electron microscopy and 3D reconstruction

Cortactin and WASP activate Arp2/3-mediated actin filament nucleation and branching. However, different mechanisms underlie activation by the two proteins, which rely on distinct actin-binding modules and modes of binding to actin filaments. It is generally thought that cortactin binds to "mother" actin filaments, while WASP donates actin monomers to Arp2/3-generated "daughter" filament branches. Interestingly, cortactin also binds WASP in addition to F-actin and the Arp2/3 complex. However, the structural basis for the role of cortactin in filament branching remains unknown, making interpretation difficult. Here, electron microscopy and 3D reconstruction were carried out on F-actin decorated with the actin-binding repeating domain of cortactin, revealing conspicuous density on F-actin attributable to cortactin that is located on a consensus-binding site on subdomain-1 of actin subunits. Strikingly, the binding of cortactin widens the gap between the two long-pitch filament strands. Although other proteins have been found to alter the structure of the filament, the cortactin-induced conformational change appears unique. The results are consistent with a mechanism whereby alterations of the F-actin structure may facilitate recruitment of the Arp2/3 complex to the "mother" filament in the cortex of cells. In addition, cortactin may act as a structural adapter protein, stabilizing nascent filament branches while mediating the simultaneous recruitment of Arp2/3 and WASP.     

Three-dimensional reconstruction of bacterial viruses from electron microscopy data. Bacteriophage structure

The structure of bacteriophages from different groups is presented based on the data obtained by the three-dimensional reconstruction, optical diffraction and filtration and electron microscopy techniques. The study made possible to suggest the scheme for the mechanism of sheath molecules rearrangement at contraction of bacteriophage tail sheath, the model of bacteriophage FI-1 connector. The structural elements for difference and relation of various bacteriophages are demonstrated.