Serial Block Face Imaging Research Articles

Intercellular pathways from the vasculature to the forming bone in the zebrafish larval caudal fin: Possible role in bone formation

The pathway of ion supply from the source to the site of bone deposition in vertebrates is thought to involve transport through the vasculature, followed by ion concentration in osteoblasts. The cells deposit a precursor mineral phase in vesicles, which are then exocytosed into the extracellular matrix. We observed that the entire skeleton of zebrafish larvae, is labelled within minutes after injection of calcein or FITC-dextran into the blood. This raised the possibility that there is an additional pathway of solute transport that can account for the rapid labelling. We used cryo-FIB-SEM serial block face imaging to reconstruct at high resolution the 3D ultrastructure of the caudal tail of the zebrafish larva. This reconstruction clearly shows that there is a continuous intercellular pathway from the artery to the forming bone, and from the forming bone to the vein. Fluorescence light microscopy shows that calcein and FITC-dextran form a reticulate network pattern in this tissue, which we attribute to the dye being present in the intercellular space. We conclude that this intercellular continuous space may be a supply route for ions, mineral and other solute or particulate material to the fast forming bone.

Quantitative analysis of plant ER architecture and dynamics

The endoplasmic reticulum (ER) is a highly dynamic polygonal membrane network composed of interconnected tubules and sheets (cisternae) that forms the first compartment in the secretory pathway involved in protein translocation, folding, glycosylation, quality control, lipid synthesis, calcium signalling, and metabolon formation. Despite its central role in this plethora of biosynthetic, metabolic and physiological processes, there is little quantitative information on ER structure, morphology or dynamics. Here we describe a software package (AnalyzER) to automatically extract ER tubules and cisternae from multi-dimensional fluorescence images of plant ER. The structure, topology, protein-localisation patterns, and dynamics are automatically quantified using spatial, intensity and graph-theoretic metrics. We validate the method against manually-traced ground-truth networks, and calibrate the sub-resolution width estimates against ER profiles identified in serial block-face SEM images. We apply the approach to quantify the effects on ER morphology of drug treatments, abiotic stress and over-expression of ER tubule-shaping and cisternal-modifying proteins.

Multi-Beam Scanning Electron Microscopy for High-Throughput Imaging in Connectomics Research.

Major progress has been achieved in recent years in three-dimensional microscopy techniques. This applies to the life sciences in general, but specifically the neuroscientific field has been a main driver for developments regarding volume imaging. In particular, scanning electron microscopy offers new insights into the organization of cells and tissues by volume imaging methods, such as serial section array tomography, serial block-face imaging or focused ion beam tomography. However, most of these techniques are restricted to relatively small tissue volumes due to the limited acquisition throughput of most standard imaging techniques. Recently, a novel multi-beam scanning electron microscope technology optimized to the imaging of large sample areas has been developed. Complemented by the commercialization of automated sample preparation robots, the mapping of larger, cubic millimeter range tissue volumes at high-resolution is now within reach. This Mini Review will provide a brief overview of the various approaches to electron microscopic volume imaging, with an emphasis on serial section array tomography and multi-beam scanning electron microscopic imaging.

Penetration model for chemical reactivation for resin-embedded green fluorescent protein imaging.

.In the so-called surface microscopy, serial block-face imaging is combined with mechanic sectioning to obtain volumetric imaging. While mapping a resin-embedded green fluorescent protein (GFP)-labeled specimen, it has been recently reported that an alkaline buffer is used to chemically reactivate the protonated GFP molecules, and thus improve the signal-to-noise ratio. In such a procedure, the image quality is highly affected by the penetration rate of a solution. We propose a reliable penetration model to describe the penetration process of the solution into the resin. The experimental results are consistent with the parameters predicted using this model. Thus, this model provides a valuable theoretical explanation and aids in optimizing the system parameters for mapping resin-embedded GFP biological samples.

Fast Homogeneous En Bloc Staining of Large Tissue Samples for Volume Electron Microscopy.

Fixation and staining of large tissue samples are critical for the acquisition of volumetric electron microscopic image datasets and the subsequent reconstruction of neuronal circuits. Efficient protocols exist for the staining of small samples, but uniform contrast is often difficult to achieve when the sample diameter exceeds a few hundred micrometers. Recently, a protocol (BROPA, brain-wide reduced-osmium staining with pyrogallol-mediated amplification) was developed that achieves homogeneous staining of the entire mouse brain but requires very long sample preparation times. By exploring modifications of this protocol we developed a substantially faster procedure, fBROPA, that allows for reliable high-quality staining of tissue blocks on the millimeter scale. Modifications of the original BROPA protocol include drastically reduced incubation times and a lead aspartate incubation to increase sample conductivity. Using this procedure, whole brains from adult zebrafish were stained within 4 days. Homogenous high-contrast staining was achieved throughout the brain. High-quality image stacks with voxel sizes of 10 × 10 × 25 nm3 were obtained by serial block-face imaging using an electron dose of ~15 e−/nm2. No obvious reduction in staining quality was observed in comparison to smaller samples stained by other state-of-the-art procedures. Furthermore, high-quality images with minimal charging artifacts were obtained from non-neural tissues with low membrane density. fBROPA is therefore likely to be a versatile and efficient sample preparation protocol for a wide range of applications in volume electron microscopy.

Multiscale Imaging Reveals the Hierarchical Organization of Fibrillin Microfibrils

Fibrillin microfibrils are evolutionarily ancient, structurally complex extracellular polymers found in mammalian elastic tissues where they endow elastic properties, sequester growth factors and mediate cell signalling; thus, knowledge of their structure and organization is essential for a more complete understanding of cell function and tissue morphogenesis. By combining multiple imaging techniques, we visualize three levels of hierarchical organization of fibrillin structure ranging from micro-scale fiber bundles in the ciliary zonule to nano-scale individual microfibrils. Serial block-face scanning electron microscopy imaging suggests that bundles of zonule fibers are bound together by circumferential wrapping fibers, which is mirrored on a shorter-length scale where individual zonule fibers are interwoven by smaller fibers. Electron tomography shows that microfibril directionality varies from highly aligned and parallel, connecting to the basement membrane, to a meshwork at the zonule fiber periphery, and microfibrils within the zonule are connected by short cross-bridges, potentially formed by fibrillin-binding proteins. Three-dimensional reconstructions of negative-stain electron microscopy images of purified microfibrils confirm that fibrillin microfibrils have hollow tubular structures with defined bead and interbead regions, similar to tissue microfibrils imaged in our tomograms. These microfibrils are highly symmetrical, with an outer ring and interwoven core in the bead and four linear prongs, each accommodating a fibrillin dimer, in the interbead region. Together these data show how a single molecular building block is organized into different levels of hierarchy from microfibrils to tissue structures spanning nano- to macro-length scales. Furthermore, the application of these combined imaging approaches has wide applicability to other tissue systems.

A Brief History of Microglial Ultrastructure: Distinctive Features, Phenotypes, and Functions Discovered Over the Past 60 Years by Electron Microscopy.

The first electron microscope was constructed in 1931. Several decades later, techniques were developed to allow the first ultrastructural analysis of microglia by transmission electron microscopy (EM). In the 50 years that followed, important roles of microglia have been identified, specifically due to the ultrastructural resolution currently available only with EM. In particular, the addition of electron-dense staining using immunohistochemical EM methods has allowed the identification of microglial cell bodies, as well as processes, which are difficult to recognize in EM, and to uncover their complex interactions with neurons and synapses. The ability to recognize neuronal, astrocytic, and oligodendrocytic compartments in the neuropil without any staining is another invaluable advantage of EM over light microscopy for studying intimate cell–cell contacts. The technique has been essential in defining microglial interactions with neurons and synapses, thus providing, among other discoveries, important insights into their roles in synaptic stripping and pruning via phagocytosis of extraneous synapses. Recent technological advances in EM including serial block-face imaging and focused-ion beam scanning EM have also facilitated automated acquisition of large tissue volumes required to reconstruct neuronal circuits in 3D at nanometer-resolution. These cutting-edge techniques which are now becoming increasingly available will further revolutionize the study of microglia across stages of the lifespan, brain regions, and contexts of health and disease. In this mini-review, we will focus on defining the distinctive ultrastructural features of microglia and the unique insights into their function that were provided by EM.

A novel imaging method for correlating 2D light microscopic data and 3D volume data based on block-face imaging

We have developed an imaging method designated as correlative light microscopy and block-face imaging (CoMBI), which contributes to improve the reliability of morphological analyses. This method can collect both the frozen sections and serial block-face images in a single specimen. The frozen section can be used for conventional light microscopic analysis to obtain 2-dimensional (2D) anatomical and molecular information, while serial block-face images can be used as 3-dimensional (3D) volume data for anatomical analysis. Thus, the sections maintain positional information in the specimen, and allows the correlation of 2D microscopic data and 3D volume data in a single specimen. The subjects can vary in size and type, and can cover most specimens encountered in biology. In addition, the required system for our method is characterized by cost-effectiveness. Here, we demonstrated the utility of CoMBI using specimens ranging in size from several millimeters to several centimeters, i.e., mouse embryos, human brainstem samples, and stag beetle larvae, and present successful correlation between the 2D light microscopic images and 3D volume data in a single specimen.

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.

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.

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.

Conductive resins improve charging and resolution of acquired images in electron microscopic volume imaging.

Recent advances in serial block-face imaging using scanning electron microscopy (SEM) have enabled the rapid and efficient acquisition of 3-dimensional (3D) ultrastructural information from a large volume of biological specimens including brain tissues. However, volume imaging under SEM is often hampered by sample charging, and typically requires specific sample preparation to reduce charging and increase image contrast. In the present study, we introduced carbon-based conductive resins for 3D analyses of subcellular ultrastructures, using serial block-face SEM (SBF-SEM) to image samples. Conductive resins were produced by adding the carbon black filler, Ketjen black, to resins commonly used for electron microscopic observations of biological specimens. Carbon black mostly localized around tissues and did not penetrate cells, whereas the conductive resins significantly reduced the charging of samples during SBF-SEM imaging. When serial images were acquired, embedding into the conductive resins improved the resolution of images by facilitating the successful cutting of samples in SBF-SEM. These results suggest that improving the conductivities of resins with a carbon black filler is a simple and useful option for reducing charging and enhancing the resolution of images obtained for volume imaging with SEM.

Rapid specimen preparation to improve the throughput of electron microscopic volume imaging for three-dimensional analyses of subcellular ultrastructures with serial block-face scanning electron microscopy.

Serial block-face imaging using scanning electron microscopy enables rapid observations of three-dimensional ultrastructures in a large volume of biological specimens. However, such imaging usually requires days for sample preparation to reduce charging and increase image contrast. In this study, we report a rapid procedure to acquire serial electron microscopic images within 1day for three-dimensional analyses of subcellular ultrastructures. This procedure is based on serial block-face with two major modifications, including a new sample treatment device and direct polymerization on the rivets, to reduce the time and workload needed. The modified procedure without uranyl acetate can produce tens of embedded samples observable under serial block-face scanning electron microscopy within 1day. The serial images obtained are similar to the block-face images acquired by common procedures, and are applicable to three-dimensional reconstructions at a subcellular resolution. Using this approach, regional immune deposits and the double contour or heterogeneous thinning of basement membranes were observed in the glomerular capillary loops of an autoimmune nephropathy model. These modifications provide options to improve the throughput of three-dimensional electron microscopic examinations, and will ultimately be beneficial for the wider application of volume imaging in life science and clinical medicine.

Morphological process of podocyte development revealed by block-face scanning electron microscopy.

Podocytes present a unique 3D architecture specialized for glomerular filtration. However, several 3D morphological aspects on podocyte development remain partially understood because they are difficult to reveal using conventional scanning electron microscopy (SEM). Here, we adopted serial block-face SEM imaging, a powerful tool for analyzing the 3D cellular ultrastructure, to precisely reveal the morphological process of podocyte development, such as the formation of foot processes. Development of foot processes gives rise to three morphological states: the primitive, immature and mature foot processes. Immature podocytes were columnar in shape and connected to each other by the junctional complex, which migrated toward the basal side of the cell. When the junctional complex was close to the basement membrane, immature podocytes started to interdigitate with primitive foot processes under the level of junctional complex. As primitive foot processes lengthened, the junctional complex moved between primitive foot processes to form immature foot processes. Finally, the junctional complex was gradually replaced by the slit diaphragm, resulting in the maturation of immature foot processes into mature foot processes. In conclusion, the developmental process of podocytes is now clearly visualized by block-face SEM imaging.

Three-dimensional SEM - the future of cell imaging.

For the past 50 or so years, the ability to obtain extensive three-dimensional information from cells has been the preserve of those brave electron microscopists willing to undertake extensive serial thin sectioning followed by manual or semiautomated reconstruction. More recently, the technique of transmission electron tomography has enabled the high-resolution study of subcellular structure at extremely high resolution in 3-D. However, to obtain reconstructions to any great depth using this technique is time-intensive, involves collecting serial tomograms, is extremely skilful and reconstructions can take many weeks to complete (Marsh, 2005). The development of high-resolution field emission scanning electron microscopes has enabled biologists to explore the use of the SEM to study cell ultrastructure in resin-embedded material using traditional TEM preparative techniques. Combined with ion beam milling (FIBSEM) techniques (Kizilyaprak et al., 2014) or the ability to undertake ultramicrotomy within the SEM specimen chamber, known as serial block face imaging (SBFSEM, SB-EM), it is now possible to collect data sets of many microns in the z-axis (Hughes et al., 2014) and reconstruct organelle organization in whole cells or tissues. This special edition of the Journal of Microscopy contains a series of papers exploring the developments and applications of the use of scanning electron microscopy to study cell ultrastructure. This includes papers on correlative fluorescence and SEM imaging, SBFSEM and FIB-SEM, through to array tomography of sections imaged by SEM and high-throughput imaging using a multibeam SEM. We thank ZEISS Microscopy for sponsoring this edition and for making all the articles freely available through Open Access.

Three-dimensional architecture of podocytes revealed by block-face scanning electron microscopy.

Block-face imaging is a scanning electron microscopic technique which enables easier acquisition of serial ultrastructural images directly from the surface of resin-embedded biological samples with a similar quality to transmission electron micrographs. In the present study, we analyzed the three-dimensional architecture of podocytes using serial block-face imaging. It was previously believed that podocytes are divided into three kinds of subcellular compartment: cell body, primary process, and foot process, which are simply aligned in this order. When the reconstructed podocytes were viewed from their basal side, the foot processes were branched from a ridge-like prominence, which was formed on the basal surface of the primary process and was similar to the usual foot processes in structure. Moreover, from the cell body, the foot processes were also emerged via the ridge-like prominence, as found in the primary process. The ridge-like prominence anchored the cell body and primary process to the glomerular basement membrane, and connected the foot processes to the cell body and primary process. In conclusion, serial block-face imaging is a powerful tool for clear understanding the three-dimensional architecture of podocytes through its ability to reveal novel structures which were difficult to determine by conventional transmission and scanning electron microscopes alone.

Focus on photonics, biomedical optics, and microscopy

Focus on photonics, biomedical optics, and microscopy

Mission (im)possible - mapping the brain becomes a reality.

Charting and understanding the full wiring diagram of complex neuronal structures such as the central nervous system or the brain (Connectomics) is one of the big remaining challenges in life sciences. Although at first it appears nearly impossible to map out a full diagram of, e.g., a mouse brain with sufficient resolution to identify each and every connection between neurons, recent technological advances move such an ambitious undertaking into the realms of possibility without spending decades at a microscope. However there are still many challenges to address in order to pave the way for fast and systematic neurobiological understanding of whole networks. These challenges range from a more robust and reproducible sample preparation to automated image data acquisition, more efficient data storage strategies and powerful data analysis tools. Here we will review novel imaging techniques developed for the challenge of mapping out the full connectome of a nervous system, brain or eye to name just a few examples. The imaging techniques reviewed cover light sheet illumination methods, single and multi-beam scanning electron microscopy, and we will briefly mention the possible combination of both light and electron microscopy. In particular we will review 'clearing' and in vivo methods that can be performed with light sheet fluroescence microscopes such as the ZEISS Lightsheet Z.1. We will then focus on scanning electron microscopy with single and multi-beam instruments including methods such as serial blockface imaging and array tomography methods.

X-Ray Microscopy as an Approach to Increasing Accuracy and Efficiency of Serial Block-Face Imaging for Correlated Light and Electron Microscopy of Biological Specimens

The recently developed three-dimensional electron microscopic (EM) method of serial block-face scanning electron microscopy (SBEM) has rapidly established itself as a powerful imaging approach. Volume EM imaging with this scanning electron microscopy (SEM) method requires intense staining of biological specimens with heavy metals to allow sufficient back-scatter electron signal and also to render specimens sufficiently conductive to control charging artifacts. These more extreme heavy metal staining protocols render specimens light opaque and make it much more difficult to track and identify regions of interest (ROIs) for the SBEM imaging process than for a typical thin section transmission electron microscopy correlative light and electron microscopy study. We present a strategy employing X-ray microscopy (XRM) both for tracking ROIs and for increasing the efficiency of the workflow used for typical projects undertaken with SBEM. XRM was found to reveal an impressive level of detail in tissue heavily stained for SBEM imaging, allowing for the identification of tissue landmarks that can be subsequently used to guide data collection in the SEM. Furthermore, specific labeling of individual cells using diaminobenzidine is detectable in XRM volumes. We demonstrate that tungsten carbide particles or upconverting nanophosphor particles can be used as fiducial markers to further increase the precision and efficiency of SBEM imaging.