Block Face Research Articles

UltraLM and miniLM: Locator tools for smart tracking of fluorescent cells in correlative light and electron microscopy.

In-resin fluorescence (IRF) protocols preserve fluorescent proteins in resin-embedded cells and tissues for correlative light and electron microscopy, aiding interpretation of macromolecular function within the complex cellular landscape. Dual-contrast IRF samples can be imaged in separate fluorescence and electron microscopes, or in dual-modality integrated microscopes for high resolution correlation of fluorophore to organelle. IRF samples also offer a unique opportunity to automate correlative imaging workflows. Here we present two new locator tools for finding and following fluorescent cells in IRF blocks, enabling future automation of correlative imaging. The ultraLM is a fluorescence microscope that integrates with an ultramicrotome, which enables 'smart collection' of ultrathin sections containing fluorescent cells or tissues for subsequent transmission electron microscopy or array tomography. The miniLM is a fluorescence microscope that integrates with serial block face scanning electron microscopes, which enables 'smart tracking' of fluorescent structures during automated serial electron image acquisition from large cell and tissue volumes.

Defining the hierarchical organisation of collagen VI microfibrils at nanometre to micrometre length scales.

Cartilage is a connective tissue rich in extracellular matrix molecules and is tough and compressive to cushion the bones of joints. However, in adults cartilage is poorly repaired after injury and so this is an important target for tissue engineering. Many connective tissues contain collagen VI, which forms microfibrils and networks but we understand very little about these assemblies or the tissue structures they form. Therefore, we have use complementary imaging techniques to image collagen VI microfibrils from the nano-scale to the micro-scale in order to understand the structure and the assemblies it forms. These findings will help to inform the future design of scaffolds to mimic connective tissues in regenerative medicine applications.

Mitochondrial growth during the cell cycle of Trypanosoma brucei bloodstream forms.

Mitochondrial organelles need to be replicated during cell division. Many aspects of this process have been studied in great detail, however the actual size increase and the position of organelle growth are less well understood. We use the protozoan parasite Trypanosoma brucei that contains a single mitochondrion to study organelle biogenesis by fluorescence microscopy. From the analysis of more than 1000 T. brucei bloodstream form cells of a nonsynchronous population we conclude that the mitochondrial network mostly grows from two areas along the main organelle axis, posterior and anterior of the nucleus. Loops and branches from these two areas eventually fuse to build a complex network. Together with the appearance of the division fold in the posterior part of the cell, pruning of the mitochondrial network and finally separation into the two daughter cells occurs. Overall organelle biogenesis is not continuous during cell growth and occurs mostly in the last part of the cell cycle. Furthermore, using 3D STED super resolution microscopy we reconstruct the volume of the organelle and characterize the region where the mitochondrial genome is positioned by serial block face scanning electron microscopy.

Broad ion beam serial section tomography

Here we examine the potential of serial Broad Ion Beam (BIB) Ar+ ion polishing as an advanced serial section tomography (SST) technique for destructive 3D material characterisation for collecting data from volumes with lateral dimensions significantly greater than 100µm and potentially over millimetre sized areas. Further, the associated low level of damage introduced makes BIB milling very well suited to 3D EBSD acquisition with very high indexing rates. Block face serial sectioning data registration schemes usually assume that the data comprises a series of parallel, planar slices. We quantify the variations in slice thickness and parallelity which can arise when using BIB systems comparing Gatan PECS and Ilion BIB systems for large volume serial sectioning and 3D-EBSD data acquisition. As a test case we obtain 3D morphologies and grain orientations for both phases of a WC-11%wt. Co hardmetal. In our case we have carried out the data acquisition through the manual transfer of the sample between SEM and BIB which is a very slow process (1–2 slice per day), however forthcoming automated procedures will markedly speed up the process. We show that irrespective of the sectioning method raw large area 2D-EBSD maps are affected by distortions and artefacts which affect 3D-EBSD such that quantitative analyses and visualisation can give misleading and erroneous results. Addressing and correcting these issues will offer real benefits when large area (millimetre sized) automated serial section BIBS is developed.

Tunneling in lesser Himalaya, Jammu and Kashmir, India with special emphasis on tectonic mélange

Himalayan fold belt has full of geological surprises, ‘melange’ is one of them which create difficulties during tunneling. Such melange completely went unnoticed during surface mapping and geotechnical investigation preceding the construction of the Udhampur railway tunnel (URT). During the construction, the melange zone has encountered across the tunnel, which occurs along the Tanhal thrust (equivalent to MBT) that separates the Murree Group and the Shiwalik Group. The melange was characterized by a chaotic, heterogeneous geological mixture of stronger blocks (scale independence) and weaker sheared fine-grained matrix, often termed as “block-in-matrix rocks” or bimrocks, which enforced mixed face tunneling. The heterogeneity in a tectonic melange led to stress concentrations in the rock blocks, and there were relatively high deformations within the matrix also. Release of stress from the blocks due to excavation, with unfavorable joint and thrust orientations enforced brittle failure of the blocks (face and crown collapses) while matrix deformation (time dependent) caused convergence of primary support later. Additionally, the clay minerals with high swelling potential within the matrix swelled and created pressure on the primary support. Due to the geomechanical heterogeneity in melange, homogenizing the rock-mass by the commonly used quantitative systems might have lead to an inappropriate design and construction. The adopted New Austrian Tunneling Method (NATM) proved to be an useful tool for tunneling.

The parasitic protozoan Trypanosoma brucei contains a non canonical protein translocase in the outer membrane of its mitochondrion. A 3D reconstruction of the insect stage form of the parasite (obtained by serial block face scanning electron microscopy) i

Molecular MicrobiologyVolume 102, Issue 3 p. i-i Front CoverFree Access The parasitic protozoan Trypanosoma brucei contains a non canonical protein translocase in the outer membrane of its mitochondrion. A 3D reconstruction of the insect stage form of the parasite (obtained by serial block face scanning electron microscopy) illustrates the network-like mitochondrion (green), basal body (white), flagellum (purple) and DNA (blue). Image courtesy: Jiří Týč and Sue Vaughan (Oxford Brookes University, Oxford, UK). For details see article by Desy et al. on pp. 520–529 of this issue. First published: 21 October 2016 https://doi.org/10.1111/mmi.13195AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume102, Issue3November 2016Pages i-i RelatedInformation

Surface Hydroxyl OH Defects of η-Al2O3 and χ-Al2O3 by Solid State NMR, XRD, and DFT Calculations

Abstract For the first time, the detailed structure of χ-Al2O3 and η-Al2O3 surface has been established by implementing the NMR crystallography approach. The surface of η-Al2O3 has been demonstrated to be formed primarily by the (111) facets, while the χ-Al2O3 surface is a combination of (111) and (110) facets. This observation supports the block model of aluminum oxides previously proposed by Tsybulya and Kryukova [S. V. Tsybulya, G. N. Kryukova, Phys. Rev. B 77 (2008) 024112.]. The additional terminal OH groups, observed experimentally and not contributing to (111) and (110) theoretical calculations, are considered to be bonded to the tetrahedral aluminum sites. Their origin is related to the junctions of crystallographic faces of spinel building blocks, being a part of discussed model. Higher content of these terminal OH groups in χ-Al2O3 is a result of more junctions in the case of its more mosaic structure compared to η-Al2O3.

Using electron microscopes to look into the lung.

In the nineteenth century, there was a dispute about the existence of a lung alveolar epithelium which remained unsolved until the invention of electron microscopy (EM) and its application to the lung. From the early 1960s, Ewald Weibel became the master of lung EM. He showed that the alveolar epithelium is covered with a lining layer containing surfactant. Weibel also explained the phenomenon of "non-nucleated plates" observed already in 1881 by Albert Kölliker. Weibel's most significant contribution was to the development of stereological methods. Therefore, quantitative characterization of lung structure revealing structure-function relationships became possible. Today, the spectrum of EM methods to study the fine structure of the lung has been extended significantly. Cryo-preparation techniques are available which are necessary for immunogold labeling of molecules. Energy-filtering techniques can be used for the detection of elements. There have also been major improvements in stereology, thus providing a very versatile toolbox for quantitative lung phenotype analyses. A new dimension was added by 3D EM techniques. Depending on the desired sample size and resolution, the spectrum ranges from array tomography via serial block face scanning EM and focused ion beam scanning EM to electron tomography. These 3D datasets provide new insights into lung ultrastructure. Biomedical EM is an ever-developing field. Its high resolution remains unparalleled. Moreover, EM has the unique advantage of providing an "open view" into cells and tissues within their full architectural context. Therefore, EM will remain an indispensable tool for a better understanding of the lung's functional design.

Cryo-FIB-SEM serial milling and block face imaging: Large volume structural analysis of biological tissues preserved close to their native state.

Many important biological questions can be addressed by studying in 3D large volumes of intact, cryo fixed hydrated tissues (⩾10,000μm3) at high resolution (5-20nm). This can be achieved using serial FIB milling and block face surface imaging under cryo conditions. Here we demonstrate the unique potential of the cryo-FIB-SEM approach using two extensively studied model systems; sea urchin embryos and the tail fin of zebrafish larvae. We focus in particular on the environment of mineral deposition sites. The cellular organelles, including mitochondria, Golgi, ER, nuclei and nuclear pores are made visible by the image contrast created by differences in surface potential of different biochemical components. Auto segmentation and/or volume rendering of the image stacks and 3D reconstruction of the skeleton and the cellular environment, provides a detailed view of the relative distribution in space of the tissue/cellular components, and thus of their interactions. Simultaneous acquisition of secondary and back-scattered electron images adds additional information. For example, a serial view of the zebrafish tail reveals the presence of electron dense mineral particles inside mitochondrial networks extending more than 20μm in depth in the block. Large volume imaging using cryo FIB SEM, as demonstrated here, can contribute significantly to the understanding of the structures and functions of diverse biological tissues.

Fluorescence imaging of cambial zones to study wood formation in Pinus radiata D. Don.

A technique whereby whole mounts of delicate tissues of differentiating xylem are imaged directly by polishing and block-face imaging of embedded microcores. Autofluorescence and image analysis aids identifying the stages of xylogenesis. Stem microcores from fast-growing trees, such as Pinus radiata (D. Don) with wide zones of cambium and differentiating xylem and very wide growth rings, pose a challenge for microscopy, as they are difficult to handle and easily damaged compared to slower growing species. A novel procedure has been developed which captures high-resolution images directly from the block face of large samples embedded in plastic resin without the need for sectioning or staining. Microcores of differentiating xylem of P. radiata growing in the central North Island of New Zealand were embedded in a low viscosity acrylic resin. The surface of the entire resin block was abraded and polished to expose cross sections of the wide zone of wood formation in these fast-growing trees without damage or distortion. Autofluorescence imaging was performed using a confocal laser scanning microscope. This avoided the need for staining and allowed the determination of the beginning of lignification based on lignin autofluorescence. Image analysis was used to determine the widths of: (a) the cambium, cell expansion, and wall-thickening zone (CET) and (b) the wall lignification zone (LT). A fast-growing tree had wider CET and LT zones than a slow-growing tree. This was due to the fast-growing tree producing more tracheids than the slow-growing tree, rather than by the production of larger tracheids.

Deceleration of probe beam by stage bias potential improves resolution of serial block-face scanning electron microscopic images.

Serial block-face scanning electron microscopy (SBEM) is quickly becoming an important imaging tool to explore three-dimensional biological structure across spatial scales. At probe-beam-electron energies of 2.0 keV or lower, the axial resolution should improve, because there is less primary electron penetration into the block face. More specifically, at these lower energies, the interaction volume is much smaller, and therefore, surface detail is more highly resolved. However, the backscattered electron yield for metal contrast agents and the backscattered electron detector sensitivity are both sub-optimal at these lower energies, thus negating the gain in axial resolution. We found that the application of a negative voltage (reversal potential) applied to a modified SBEM stage creates a tunable electric field at the sample. This field can be used to decrease the probe-beam-landing energy and, at the same time, alter the trajectory of the signal to increase the signal collected by the detector. With decelerated low landing-energy electrons, we observed that the probe-beam-electron-penetration depth was reduced to less than 30 nm in epoxy-embedded biological specimens. Concurrently, a large increase in recorded signal occurred due to the re-acceleration of BSEs in the bias field towards the objective pole piece where the detector is located. By tuning the bias field, we were able to manipulate the trajectories of the primary and secondary electrons, enabling the spatial discrimination of these signals using an advanced ring-type BSE detector configuration or a standard monolithic BSE detector coupled with a blocking aperture.

Corrosion Protection of Zinc Alloy Coated Steel By Organic Coatings Pigmented with Strontium Aluminium Polyphosphate

Strontium aluminium polyphosphate (SAPP) inhibitive pigment formulated into an organic primer for corrosion protection of zinc alloy coated steel in 0.6 M sodium chloride solution was investigated by serial block face imaging using scanning electron microscopy, which provided detailed information on inhibitor releasing, transportation and reaction with zinc alloy substrate. It was found that SAPP contains at least two distinct particle types, one which is strontium rich and another which is aluminium rich. These particles have different release characteristics which are dependent on the local environment. The strontium rich particles release their components faster in near neutral and acidic environment compared with the aluminium rich particles whilst in an alkaline environment the reverse is found. The pH related inhibitor release process and the mixture of the inhibitive pigment with different strontium to aluminium ratios ensure the availability of the inhibitor at both anodic and cathodic locations, which consequently reduced/retarded the corrosion of zinc alloy coated steel.

High resolution three dimensional imaging of placental villi using serial block face scanning electron microscopy

High resolution three dimensional imaging of placental villi using serial block face scanning electron microscopy

The Spectrum of Mitochondrial Ultrastructural Defects in Mitochondrial Myopathy.

Mitochondrial functions are intrinsically linked to their morphology and membrane ultrastructure. Characterizing abnormal mitochondrial structural features may thus provide insight into the underlying pathogenesis of inherited and acquired mitochondrial diseases. Following a systematic literature review on ultrastructural defects in mitochondrial myopathy, we investigated skeletal muscle biopsies from seven subjects with genetically defined mtDNA mutations. Mitochondrial ultrastructure and morphology were characterized using two complimentary approaches: transmission electron microscopy (TEM) and serial block face scanning EM (SBF-SEM) with 3D reconstruction. Six ultrastructural abnormalities were identified including i) paracrystalline inclusions, ii) linearization of cristae and abnormal angular features, iii) concentric layering of cristae membranes, iv) matrix compartmentalization, v) nanotunelling, and vi) donut-shaped mitochondria. In light of recent molecular advances in mitochondrial biology, these findings reveal novel aspects of mitochondrial ultrastructure and morphology in human tissues with implications for understanding the mechanisms linking mitochondrial dysfunction to disease.

Effects of facing, reinforcement stiffness, toe resistance, and height on reinforced walls

This study numerically investigated the combined effect of reinforcement and facing stiffness, wall height, and toe resistance on the behavior of reinforced soil (RS) walls under working stress conditions. For RS walls with vertical segmental block facing, parametric analyses showed that the combined effect of the facing stiffness, wall height, and toe resistance on the distribution of the maximum reinforcement load with depth may be limited to approximately 4 m above the base of the wall. Furthermore, the shape of the distribution of the reinforcement load may be a function of the combined effect of the wall height, reinforcement stiffness, toe resistance, and facing stiffness. For a given facing stiffness and fixed-base conditions, increasing the height of the wall and reinforcement stiffness may change the distribution shape of the reinforcement load from trapezoidal to the triangular. Additionally, the maximum reinforcement loads calculated using finite element analyses were compared to the values predicted by design methods found in the literature. Some limitations of those design procedures are presented and discussed.

Analysis of Mitochondrial Networks by Serial Block Face SEM

Analysis of Mitochondrial Networks by Serial Block Face SEM

Three-dimensional visualisation of soft biological structures by X-ray computed micro-tomography.

Whereas the two-dimensional (2D) visualisation of biological samples is routine, three-dimensional (3D) imaging remains a time-consuming and relatively specialised pursuit. Current commonly adopted techniques for characterising the 3D structure of non-calcified tissues and biomaterials include optical and electron microscopy of serial sections and sectioned block faces, and the visualisation of intact samples by confocal microscopy or electron tomography. As an alternative to these approaches, X-ray computed micro-tomography (microCT) can both rapidly image the internal 3D structure of macroscopic volumes at sub-micron resolutions and visualise dynamic changes in living tissues at a microsecond scale. In this Commentary, we discuss the history and current capabilities of microCT. To that end, we present four case studies to illustrate the ability of microCT to visualise and quantify: (1) pressure-induced changes in the internal structure of unstained rat arteries, (2) the differential morphology of stained collagen fascicles in tendon and ligament, (3) the development of Vanessa cardui chrysalises, and (4) the distribution of cells within a tissue-engineering construct. Future developments in detector design and the use of synchrotron X-ray sources might enable real-time 3D imaging of dynamically remodelling biological samples.

Serial block face scanning electron microscopy and the reconstruction of plant cell membrane systems.

Serial block face imaging with the scanning electron microscope has been developed as an alternative to serial sectioning and transmission electron microscopy for the ultrastructural analysis of the three-dimensional organization of cells and tissues. An ultramicrotome within the microscope specimen chamber permits sectioning and imaging to a depth of many microns within resin-embedded specimens. The technology has only recently been adopted by plant microscopists and here we describe some specimen preparation procedures suitable for plant tissue, suggested microscope imaging parameters and discuss the software required for image reconstruction and analysis.

A methodological pipeline for serial-section imaging and tissue realignment for whole-brain functional and connectivity assessment

BackgroundUnderstanding the neurobiological basis of cognition and behavior, and disruptions to these processes following injury and disease, requires a large-scale assessment of neural populations, and knowledge of their patterns of connectivity. New methodWe present an analysis platform for large-scale investigation of functional and neuroanatomical connectivity in rodents. Retrograde tracers were injected and in a subset of animals behavioral tests to drive immediate-early gene expression were administered. This approach allows users to perform whole-brain assessment of function and connection in a semi-automated quantitative manner. Brains were cut in the coronal plane, and an image of the block face was acquired. Wide-field fluorescent scans of whole sections were acquired and analyzed using Matlab software. ResultsThe toolkit utilized open-source and custom platforms to accommodate a largely automated analysis pipeline in which neuronal boundaries are automatically segmented, the position of segmented neurons are co-registered with a corresponding image acquired during sectioning, and a 3-D representation of neural tracer (and other products) throughout the entire brain is generated. Comparison with existing methodsCurrent whole brain connectivity measures primarily target mice and use anterograde tracers. Our focus on segmented units of interest (e.g., NeuN labeled neurons) and restricting measures to these units produces a flexible platform for a variety of whole brain analyses (measuring activation, connectivity, markers of disease, etc.). ConclusionsThis open-source toolkit allows an investigator to visualize and quantify whole brain data in 3-D, and additionally provides a framework that can be rapidly integrated with user-specific analyses and methodologies.

Microscopic validation of whole mouse micro-metastatic tumor imaging agents using cryo-imaging and sliding organ image registration.

We created a metastasis imaging, analysis platform consisting of software and multi-spectral cryo-imaging system suitable for evaluating emerging imaging agents targeting micro-metastatic tumor. We analyzed CREKA-Gd in MRI, followed by cryo-imaging which repeatedly sectioned and tiled microscope images of the tissue block face, providing anatomical bright field and molecular fluorescence, enabling 3D microscopic imaging of the entire mouse with single metastatic cell sensitivity. To register MRI volumes to the cryo bright field reference, we used our standard mutual information, non-rigid registration which proceeded: preprocess → affine → B-spline non-rigid 3D registration. In this report, we created two modified approaches: mask where we registered locally over a smaller rectangular solid, and sliding organ. Briefly, in sliding organ, we segmented the organ, registered the organ and body volumes separately and combined results. Though sliding organ required manual annotation, it provided the best result as a standard to measure other registration methods. Regularization parameters for standard and mask methods were optimized in a grid search. Evaluations consisted of DICE, and visual scoring of a checkerboard display. Standard had accuracy of 2 voxels in all regions except near the kidney, where there were 5 voxels sliding. After mask and sliding organ correction, kidneys sliding were within 2 voxels, and Dice overlap increased 4%-10% in mask compared to standard. Mask generated comparable results with sliding organ and allowed a semi-automatic process.