Dimensional Electron Microscopy Research Articles

Using Scipion for stream image processing at Cryo-EM facilities

Three dimensional electron microscopy is becoming a very data-intensive field in which vast amounts of experimental images are acquired at high speed. To manage such large-scale projects, we had previously developed a modular workflow system called Scipion (de la Rosa-Trevín et al., 2016). We present here a major extension of Scipion that allows processing of EM images while the data is being acquired. This approach helps to detect problems at early stages, saves computing time and provides users with a detailed evaluation of the data quality before the acquisition is finished. At present, Scipion has been deployed and is in production mode in seven Cryo-EM facilities throughout the world.

Novel Microscopic Techniques for Podocyte Research.

Together with endothelial cells and the glomerular basement membrane, podocytes form the size-specific filtration barrier of the glomerulus with their interdigitating foot processes. Since glomerulopathies are associated with so-called foot process effacement—a severe change of well-formed foot processes into flat and broadened processes—visualization of the three-dimensional podocyte morphology is a crucial part for diagnosis of nephrotic diseases. However, interdigitating podocyte foot processes are too narrow to be resolved by classic light microscopy due to Ernst Abbe's law making electron microscopy necessary. Although three dimensional electron microscopy approaches like serial block face and focused ion beam scanning electron microscopy and electron tomography allow volumetric reconstruction of podocytes, these techniques are very time-consuming and too specialized for routine use or screening purposes. During the last few years, different super-resolution microscopic techniques were developed to overcome the optical resolution limit enabling new insights into podocyte morphology. Super-resolution microscopy approaches like three dimensional structured illumination microscopy (3D-SIM), stimulated emission depletion microscopy (STED) and localization microscopy [stochastic optical reconstruction microscopy (STORM), photoactivated localization microscopy (PALM)] reach resolutions down to 80–20 nm and can be used to image and further quantify podocyte foot process morphology. Furthermore, in vivo imaging of podocytes is essential to study the behavior of these cells in situ. Therefore, multiphoton laser microscopy was a breakthrough for in vivo studies of podocytes in transgenic animal models like rodents and zebrafish larvae because it allows imaging structures up to several hundred micrometer in depth within the tissue. Additionally, along with multiphoton microscopy, lightsheet microscopy is currently used to visualize larger tissue volumes and therefore image complete glomeruli in their native tissue context. Alongside plain visualization of cellular structures, atomic force microscopy has been used to study the change of mechanical properties of podocytes in diseased states which has been shown to be a culprit in podocyte maintenance. This review discusses recent advances in the field of microscopic imaging and demonstrates their currently used and other possible applications for podocyte research.

Three dimensional electron microscopy reveals changing axonal and myelin morphology along normal and partially injured optic nerves

Following injury to the central nervous system, axons and myelin distinct from the initial injury site undergo changes associated with compromised function. Quantifying such changes is important to understanding the pathophysiology of neurotrauma; however, most studies to date used 2 dimensional (D) electron microscopy to analyse single sections, thereby failing to capture changes along individual axons. We used serial block face scanning electron microscopy (SBF SEM) to undertake 3D reconstruction of axons and myelin, analysing optic nerves from normal uninjured female rats and following partial optic nerve transection. Measures of axon and myelin dimensions were generated by examining 2D images at 5 µm intervals along the 100 µm segments. In both normal and injured animals, changes in axonal diameter, myelin thickness, fiber diameter, G-ratio and percentage myelin decompaction were apparent along the lengths of axons to varying degrees. The range of values for axon diameter along individual reconstructed axons in 3D was similar to the range from 2D datasets, encompassing reported variation in axonal diameter attributed to retinal ganglion cell diversity. 3D electron microscopy analyses have provided the means to demonstrate substantial variability in ultrastructure along the length of individual axons and to improve understanding of the pathophysiology of neurotrauma.

Correlative Fluorescence and Electron Microscopy in 3D-Scanning Electron Microscope Perspective.

The ability to correlate fluorescence microscopy (FM) and electron microscopy (EM) data obtained on biological (cell and tissue) specimens is essential to bridge the resolution gap between the data obtained by these different imaging techniques. In the past such correlations were limited to either EM navigation in two dimensions to the locations previously highlighted by fluorescence markers, or subsequent high-resolution acquisition of tomographic information using a TEM. We present a novel approach whereby a sample previously investigated by FM is embedded and subjected to sequential mechanical polishing and backscatter imaging by scanning electron microscope. The resulting three dimensional EM tomogram of the sample can be directly correlated to the FM data. © 2017 by John Wiley & Sons, Inc.

Resolution of the three dimensional structure of components of the glomerular filtration barrier

BackgroundThe human glomerulus is the primary filtration unit of the kidney, and contains the Glomerular Filtration Barrier (GFB). The GFB had been thought to comprise 3 layers – the endothelium, the basement membrane and the podocyte foot processes. However, recent studies have suggested that at least two additional layers contribute to the function of the GFB, the endothelial glycocalyx on the vascular side, and the sub-podocyte space on the urinary side. To investigate the structure of these additional layers is difficult as it requires three-dimensional reconstruction of delicate sub-microscopic (<1 μm) cellular and extracellular elements.MethodsHere we have combined three different advanced electron microscopic techniques that cover multiple orders of magnitude of volume sampled, with a novel staining methodology (Lanthanum Dysprosium Glycosaminoglycan adhesion, or LaDy GAGa), to determine the structural basis of these two additional layers. Serial Block Face Scanning Electron Microscopy (SBF-SEM) was used to generate a 3-D image stack with a volume of a 5.3 x 105 μm3 volume of a whole kidney glomerulus (13% of glomerular volume). Secondly, Focused Ion Beam milling Scanning Electron Microscopy (FIB-SEM) was used to image a filtration region (48 μm3 volume). Lastly Transmission Electron Tomography (Tom-TEM) was performed on a 0.3 μm3 volume to identify the fine structure of the glycocalyx.ResultsTom-TEM clearly showed 20 nm fibre spacing in the glycocalyx, within a limited field of view. FIB-SEM demonstrated, in a far greater field of view, how the glycocalyx structure related to fenestrations and the filtration slits, though without the resolution of TomTEM. SBF-SEM was able to determine the extent of the sub-podocyte space and glycocalyx coverage, without additional heavy metal staining. Neither SBF- nor FIB-SEM suffered the anisotropic shrinkage under the electron beam that is seen with Tom-TEM.ConclusionsThese images demonstrate that the three dimensional structure of the GFB can be imaged, and investigated from the whole glomerulus to the fine structure of the glycocalyx using three dimensional electron microscopy techniques. This should allow the identification of structural features regulating physiology, and their disruption in pathological states, aiding the understanding of kidney disease.

Lis1 Regulates Dynein as a Molecular Wedge

Cytoplasmic dynein walks along microtubules by coupling cycles of ATP hydrolysis in its motor domain with cycles of microtubule binding and release at its microtubule-binding domain. Unlike the other cytoskeletal motors--kinesin and myosin--that have achieved functional diversity through molecular diversity, a single isoform of cytoplasmic dynein is responsible for a host of cellular functions. These range from transport of macromolecular cargo to the construction of the mitotic spindle. How is dynein tuned to perform these different tasks? Regulation is a likely source of functional versatility and understanding it is an area of intense focus in the motors field.Lis1 is one of dynein's essential and ubiquitous regulators. We have previously shown that Lis1 acts as a molecular “clutch”, disengaging the cycles of ATP hydrolysis from the cycles of microtubule binding and release. This results in a dynein-Lis1 complex that remains bound to a microtubule despite continuing ATP hydrolysis. We also showed, using negative stain EM and 2D image analysis, that Lis1 binds to dynein's motor domain at AAA3/4.We have now used a combination of three dimensional electron microscopy, biochemistry and single molecule methods to characterize the mechanism by which Lis1 regulates dynein. A cryo-negative stain structure of the dynein-Lis1 complex (21A resolution) shows that Lis1 physically blocks dynein's linker domain from docking at its normal AAA5 site under no nucleotide conditions. Instead, the N-terminal half of the linker takes an alternative path around Lis1. Our single molecule data show that a shortened linker, short enough to bypass Lis1 but long enough to preserve its interaction with AAA5, renders dynein insensitive to Lis1. Taken together, our data suggest that Lis1 achieves its uncoupling activity by acting as a molecular wedge.

Three dimensional electron microscopy and in silico tools for macromolecular structure determination.

Recently, structural biology witnessed a major tool - electron microscopy - in solving the structures of macromolecules in addition to the conventional techniques, X-ray crystallography and nuclear magnetic resonance (NMR). Three dimensional transmission electron microscopy (3DTEM) is one of the most sophisticated techniques for structure determination of molecular machines. Known to give the 3-dimensional structures in its native form with literally no upper limit on size of the macromolecule, this tool does not need the crystallization of the protein. Combining the 3DTEM data with in silico tools, one can have better refined structure of a desired complex. In this review we are discussing about the recent advancements in three dimensional electron microscopy and tools associated with it.

Progressive sheet-to-tubule transformation is a general mechanism for endoplasmic reticulum partitioning in dividing mammalian cells

The endoplasmic reticulum (ER) is both structurally and functionally complex, consisting of a dynamic network of interconnected sheets and tubules. To achieve a more comprehensive view of ER organization in interphase and mitotic cells and to address a discrepancy in the field (i.e., whether ER sheets persist, or are transformed to tubules, during mitosis), we analyzed the ER in four different mammalian cell lines using live-cell imaging, high-resolution electron microscopy, and three dimensional electron microscopy. In interphase cells, we found great variation in network organization and sheet structures among different cell lines. In mitotic cells, we show that the ER undergoes both spatial reorganization and structural transformation of sheets toward more fenestrated and tubular forms. However, the extent of spatial reorganization and sheet-to-tubule transformation varies among cell lines. Fenestration and tubulation of the ER correlates with a reduced number of membrane-bound ribosomes.

Automating the Visualization of Biological Machines

With continued advances in three dimensional Electron Microscopy one is progressively able to elucidate and visualize natureˈs biological molecular machines (e.g. ribosomes, viruses) at varying resolutions and different conformations. I shall first discuss a suite of geometric optimization methods to automatically reconstruct a 3D volumetric model from a set of 2D images of the same object but with unknown pose. The solution to the inverse problem utilizes a pose determination algorithm, variational reconstruction of 3D scalar functions from 2D images, and geometry/image processing techniques of structure interpretation, feature extraction, and model refinement at progressively finer resolutions. Next, Ill briefly discuss molecular modeling techniques that enable dynamics, and visualization of structure‐function relationships of these biological machines.

Single-particle reconstruction using L(2)-gradient flow.

In this paper, we present an iterative algorithm for reconstructing a three-dimensional density function from a set of two dimensional electron microscopy images. By minimizing an energy functional consisting of a fidelity term and a regularization term, an L(2)-gradient flow is derived. The flow is integrated by a finite element method in the spatial direction and an explicit Euler scheme in the temporal direction. Our method compares favorably with those of the weighted back projection, Fourier method, algebraic reconstruction technique and simultaneous iterative reconstruction technique.

Classification of Projections in Single Particle Electron Microscopy using Common Line Similarity Measure

In single-particle reconstruction methods [1], projections of macromolecules lying in randomly unknown orientations are collected by a transmission electron microscope. Often, several classes of conformations or binding states coexist in the sample. To obtain structures with high accuracy, it is required to separate the classes before reconstruction of the molecules. In this work, we use a graph-theoretic criterion based on common lines, which measures both the dissimilarity between the classes and the similarity within the classes. Projections are then sorted by optimizing this measure, without need for a reference volume or for any intermediate reconstructions, as done in most classification methods.The usefulness of this type of approach, via a combinatorial optimization of the measure, was first demonstrated in [2], but tested only on simulated projection data. We instead view the optimization as an eigen-decomposition problem, which makes it easy to account for different kinds of normalization of the measure. The work in [3] also uses eigen-decomposition, but the similarity is measured between 2D averages, and a reference volume is required to pre-sort the projections according to their angular orientation.In this work we measure the similarity between the projection data themselves and show the results for both simulated and experimental data.[1] J. Frank, Three Dimensional Electron Microscopy of Macromolecular Assemblies, Oxford, 2006[2] G.T. Herman and M. Kalinowski, Classification of heterogeneous electron microscopic projections into homogeneous subsets, Ultramicroscopy 108:327-338, 2008[3] M. Shatsky et al., Automated multi-model reconstruction from single-particle electron microscopy data, J. Struct. Biol. 170:98-108, 2010

On Bootstrap Techniques For Classifying Projections in Single Particle Electron Microscopy

In single-particle reconstruction methods [1], projections of macromolecules at randomly unknown orientations are collected by an electron microscope. Often, several classes of conformations or binding states coexist in a sample. To obtain structures with high accuracy, it is required to separate the classes before reconstruction of the structures takes place. In this work, we take a close look at bootstrap techniques for classifying the projection data.In the bootstrap techniques for variance estimation [2], the projection images or particles are randomly sampled with replacement from the dataset and a bootstrap volume is reconstructed from each sample, assuming known orientations. In a recent extension of the bootstrap technique to classification [3], each particle is assigned to a volume in the space spanned by the bootstrap volumes, such that the projection of the assigned volume (in the same orientation as the particle) best matches the particle. Finally, a clustering algorithm applied to the assigned volumes determines the class to which the particle belongs.In this work we explain the rational of these techniques by discussing the nature of the bootstrap volumes: i.e., how they relate to the underlying structural classes. Furthermore, several statistical analyses become easy to study in our framework. Finally, the way the particles are assigned to volumes in the space spanned by the bootstrap volumes is closely examined.[1] J. Frank, Three Dimensional Electron Microscopy of Macromolecular Assemblies, Oxford, 2006.[2] P.A. Penczek et al., Estimation of variance in single-particle reconstruction using the bootstrap technique., 154(2):168-83, JSB, 2006.[3] P.A. Penczek, Personal Communication.

Flexible fitting in 3D-EM with incomplete data on superfamily variability

We present a substantial improvement of S-flexfit, our recently proposed method for flexible fitting in three dimensional electron microscopy (3D-EM) at a resolution range of 8–12 Å, together with a comparison of the method capabilities with Normal Mode Analysis (NMA), application examples and a user’s guide. S-flexfit uses the evolutionary information contained in protein domain databases like CATH, by means of the structural alignment of the elements of a protein superfamily. The added development is based on a recent extension of the Singular Value Decomposition (SVD) algorithm specifically designed for situations with missing data: Incremental Singular Value Decomposition (ISVD). ISVD can manage gaps and allows considering more aminoacids in the structural alignment of a superfamily, extending the range of application and producing better models for the fitting step of our methodology. Our previous SVD-based flexible fitting approach can only take into account positions with no gaps in the alignment, being appropriate when the superfamily members are relatively similar and there are few gaps. However, with new data coming from structural proteomics works, the later situation is becoming less likely, making ISVD the technique of choice for further works. We present the results of using ISVD in the process of flexible fitting with both simulated and experimental 3D-EM maps (GroEL and Poliovirus 135S cell entry intermediate).

Expression of androgen receptor in X-linked spinal and bulbar muscular atrophy and amyotrophic lateral sclerosis.

<h3>Abstract</h3> The nuclear envelope has to be reformed after mitosis to create viable daughter cells with closed nuclei. How membrane sealing of DNA and assembly of nuclear pore complexes (NPCs) are achieved and coordinated is poorly understood. Here, we reconstructed nuclear membrane topology and structure of assembling NPCs in a correlative three dimensional electron microscopy time-course of dividing human cells. Our quantitative ultrastructural analysis shows that nuclear membranes form from highly fenestrated ER sheets, whose shrinking holes are stabilized and then dilated into NPCs during inner ring complex assembly, forming thousands of transport channels within minutes. This mechanism is fundamentally different from interphase NPC assembly and explains how mitotic cells can rapidly establish a closed nuclear compartment while making it transport-competent at the same time.

Myelinated nerve fibers: computed effect of myelin thickness on conduction velocity.

Myelinated nerve fibers: computed effect of myelin thickness on conduction velocity.

CONDUCTION VELOCITY AND DIAMETER OF NERVE FIBERS

ArticleCONDUCTION VELOCITY AND DIAMETER OF NERVE FIBERSJ. B. HurshJ. B. HurshFrom the Laboratories of The Rockefeller Institute for Medical Research, New YorkPublished Online:31 Jul 1939https://doi.org/10.1152/ajplegacy.1939.127.1.131MoreSectionsPDF (1005 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmail Previous Back to Top Next FiguresReferencesRelatedInformationCited ByAssessment of axonal recruitment using model-guided preclinical spinal cord stimulation in the ex vivo adult mouse spinal cordShaquia Idlett, Mallika Halder, Tianhe Zhang, Jorge Quevedo, Natalie Brill, Wendy Gu, Michael Moffitt, and Shawn Hochman24 September 2019 | Journal of Neurophysiology, Vol. 122, No. 4Three dimensional electron microscopy reveals changing axonal and myelin morphology along normal and partially injured optic nerves5 March 2018 | Scientific Reports, Vol. 8, No. 1Frequency-dependent antidromic activation in thalamocortical relay neurons: effects of synaptic inputs2 July 2018 | Journal of Neural Engineering, Vol. 15, No. 5Model-Based Vestibular Afferent Stimulation: Evaluating Selective Electrode Locations and Stimulation Waveform Shapes30 August 2018 | Frontiers in Neuroscience, Vol. 12Model-based deconstruction of cortical evoked potentials generated by subthalamic nucleus deep brain stimulationKarthik Kumaravelu,* Chintan S. Oza,* Christina E. Behrend, and Warren M. Grill9 August 2018 | Journal of Neurophysiology, Vol. 120, No. 2Model based optimal multipolar stimulation without a priori knowledge of nerve structure: application to vagus nerve stimulation23 May 2018 | Journal of Neural Engineering, Vol. 15, No. 4Specificity and robustness of long-distance connections in weighted, interareal connectomes8 May 2018 | Proceedings of the National Academy of Sciences, Vol. 115, No. 21Abnormal cardiovascular response to exercise in hypertension: contribution of neural factorsJere H. Mitchell1 June 2017 | American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, Vol. 312, No. 6Models of utricular bouton afferents: role of afferent-hair cell connectivity in determining spike train regularityWilliam R. Holmes, Janice A. Huwe, Barbara Williams, Michael H. Rowe, and Ellengene H. Peterson1 May 2017 | Journal of Neurophysiology, Vol. 117, No. 5Axon diameters and conduction velocities in the macaque pyramidal tractL. Firmin, P. Field, M. A. Maier, A. Kraskov, P. A. Kirkwood, K. Nakajima, R. N. Lemon, and M. Glickstein15 September 2014 | Journal of Neurophysiology, Vol. 112, No. 6Antidromic Activation Reveals Tonotopically Organized Projections From Primary Auditory Cortex to the Central Nucleus of the Inferior Colliculus in Guinea PigHubert H. Lim, and David J. Anderson1 February 2007 | Journal of Neurophysiology, Vol. 97, No. 2Muscle activity in the leg is tuned in response to ground reaction forcesJames M. Wakeling, Vinzenz Von Tscharner, Benno M. Nigg, and Pro Stergiou1 September 2001 | Journal of Applied Physiology, Vol. 91, No. 3Spiral nerve cuff electrode for recordings of respiratory outputMesut Sahin, Musa A. Haxhiu, Dominique M. Durand, and Ismail A. Dreshaj1 July 1997 | Journal of Applied Physiology, Vol. 83, No. 1Ischemic conduction failure and energy metabolism in experimental diabetic neuropathyP. A. Low, K. Ward, J. D. Schmelzer, and S. Brimijoin1 April 1985 | American Journal of Physiology-Endocrinology and Metabolism, Vol. 248, No. 4Afferent discharges from venous pressoreceptors in liverA. Nijima1 January 1977 | American Journal of Physiology-Cell Physiology, Vol. 232, No. 1A comparison of aortic baroreceptor discharge in normotensive and spontaneously hypertensive rats.Circulation Research, Vol. 39, No. 4 More from this issue > Volume 127Issue 1July 1939Pages 131-139 Copyright & PermissionsCopyright © 1939 by American Physiological Societyhttps://doi.org/10.1152/ajplegacy.1939.127.1.131History Received 17 May 1939 Published online 31 July 1939 Published in print 31 July 1939 PDF download Metrics Downloaded 6,360 times