Resin comparison for serial block face scanning volume electron microscopy.

Serial Block Face Scanning Electron Microscopy (SBF-SEM) is one of several volume electron microscopy (vEM) techniques whose purpose is to reveal the nanostructure of cells and tissues in three dimensions. As one of the earliest, and possibly most widely adopted of the disruptive vEM techniques there have been hundreds of publications using the method, although very few comparative studies of specimen preparation parameters. While some studies have focused on staining and specimen acquisition no comparison of resin embedding has yet been conducted. To this end we have surveyed the SBF-SEM literature to determine which resins are commonly used and compared them in both cellular and fixed tissue samples in an attempt to optimize sample preparation for: effectiveness of resin infiltration, resistance to charging and beam damage and clarity of image in the resulting data set. Here we present the results and discuss the various factors that go into optimizing specimen preparation for SBF-SEM.

Correlative light and volume electron microscopy (vCLEM): How community participation can advance developing technologies.

Correlative light and electron microscopy is a valuable tool to image samples across resolution scales and link data on structure and function. While studies using this technique have been available since the 1960s, recent developments have enabled applying these workflows to large volumes of cells and tissues. Much of the development in this area has been facilitated through the collaborative efforts of microscopists and commercial companies to bring the methods, hardware and image processing technologies needed into laboratories and core imaging facilities. This is a prime example of how what was once a niche area can be brought into the mainstream of microscopy by the efforts of imaging pioneers who push the boundaries of possibility.

Three-dimensional ultrastructure of the brain pericyte-endothelial interface.

Pericytes and endothelial cells share membranous interdigitations called "peg-and-socket" interactions that facilitate their adhesion and biochemical crosstalk during vascular homeostasis. However, the morphology and distribution of these ultrastructures have remained elusive. Using a combination of 3D electron microscopy techniques, we examined peg-and-socket interactions in mouse brain capillaries. We found that pegs extending from pericytes to endothelial cells were morphologically diverse, exhibiting claw-like morphologies at the edge of the cell and bouton-shaped swellings away from the edge. Reciprocal endothelial pegs projecting into pericytes were less abundant and appeared as larger columnar protuberances. A large-scale 3D EM data set revealed enrichment of both pericyte and endothelial pegs around pericyte somata. The ratio of pericyte versus endothelial pegs was conserved among the pericytes examined, but total peg abundance was heterogeneous across cells. These data show considerable investment between pericytes and endothelial cells, and provide morphological evidence for pericyte somata as sites of enriched physical and biochemical interaction.

Targeted Studies Using Serial Block Face and Focused Ion Beam Scan Electron Microscopy.

This protocol allows for the efficient and effective imaging of cell or tissue samples in three dimensions at the resolution level of electron microscopy. For many years electron microscopy (EM) has remained an inherently two-dimensional technique. With the advent of serial scanning electron microscope imaging techniques (volume EM), using either an integrated microtome or focused ion beam to slice then view embedded tissues, the third dimension becomes easily accessible. Serial block face scanning electron microscopy (SBF-SEM) uses an ultramicrotome enclosed in the SEM chamber. It has the capability to handle large specimens (1,000 µm x 1,000 µm) and image large fields of view at small X,Y pixel size, but is limited in the Z dimension by the diamond knife. Focused ion beam SEM (FIB-SEM) is not limited in 3D resolution, (isotropic voxels of ≤5 nm are achievable), but the field of view is much more limited. This protocol demonstrates a workflow for combining the two techniques to allow for finding individual regions of interest (ROIs) in a large field and then imaging the subsequent targeted volume at high isotropic voxel resolution. Preparing fixed cells or tissues is more demanding for volume EM techniques due to the extra contrasting needed for efficient signal generation in SEM imaging. Such protocols are time consuming and labor intensive. This protocol also incorporates microwave assisted tissue processing facilitating the penetration of reagents, which reduces the time needed for the processing protocol from days to hours.

Serial block face-scanning electron microscopy for volume electron microscopy.

There are different technologies that can be used to obtain a 3D image at nanometer resolution. Over the past decade, there has been a growing interest in applying Serial Block Face Scanning Electron Microscopy (SBF-SEM) in different fields of life science research. This technology has the advantage that it can cover a range of volumes, going from monolayers to multiple tissue layers in all three dimensions. SBF-SEM was originally used in neuroscience and then expanded to other research domains. The whole process of sample preparation for SBF-SEM is very long and consists of many steps, which makes adjustment of a given workflow very challenging. Here we describe the SBF-SEM workflow and those steps in the process that can be tweaked for any sample.

Combining serial block face and focused ion beam scanning electron microscopy for 3D studies of rare events.

Volume electron microscopy allows for the automated acquisition of serial-section imaging data that can be reconstructed in three-dimensions (3D) to provide a detailed, geometrically accurate view of cellular ultrastructure. Two, volume electron microscopy (EM) techniques, serial block face scanning electron microscopy (SBF-SEM) and focused ion beam scanning electron microscopy (FIB-SEM), use a similar slice-and-view approach but differ in their fields of view and 3D resolution. This chapter highlights a workflow where the ability of SBF-SEM to image a large field of view is combined with the precise sectioning capability of FIB-SEM to first locate a rare cellular event in a large tissue volume and then inspect the event with higher resolution. Using these two EM platforms in synergy is a powerful technique and can be useful for both simple structural studies as well as correlative studies using both light and electron microscopy.

Image Degradation in Microscopic Images: Avoidance, Artifacts, and Solutions.

The goal of modern microscopy is to acquire high-quality image based data sets. A typical microscopy workflow is set up in order to address a specific biological question and involves different steps. The first step is to precisely define the biological question, in order to properly come to an experimental design for sample preparation and image acquisition. A better object representation allows biological users to draw more reliable scientific conclusions. Image restoration can manipulate the acquired data in an effort to reduce the impact of artifacts (spurious results) due to physical and technical limitations, resulting in a better representation of the object of interest. However, precise usage of these algorithms is necessary so as to avoid further artifacts that might influence the data analysis and bias the conclusions. It is essential to understand image acquisition, and how it introduces artifacts and degradations in the acquired data, so that their effects on subsequent analysis can be minimized. This paper provides an overview of the fundamental artifacts and degradations that affect many micrographs. We describe why artifacts appear, in what sense they impact overall image quality, and how to mitigate them by first improving the acquisition parameters and then applying proper image restoration techniques.

Investigating CNS synaptogenesis at single-synapse resolution by combining reverse genetics with correlative light and electron microscopy.

Determining direct synaptic connections of specific neurons in the central nervous system (CNS) is a major technical challenge in neuroscience. As a corollary, molecular pathways controlling developmental synaptogenesis in vivo remain difficult to address. Here, we present genetic tools for efficient and versatile labeling of organelles, cytoskeletal components and proteins at single-neuron and single-synapse resolution in Drosophila mechanosensory (ms) neurons. We extended the imaging analysis to the ultrastructural level by developing a protocol for correlative light and 3D electron microscopy (3D CLEM). We show that in ms neurons, synaptic puncta revealed by genetically encoded markers serve as a reliable indicator of individual active zones. Block-face scanning electron microscopy analysis of ms axons revealed T-bar-shaped dense bodies and other characteristic ultrastructural features of CNS synapses. For a mechanistic analysis, we directly combined the single-neuron labeling approach with cell-specific gene disruption techniques. In proof-of-principle experiments we found evidence for a highly similar requirement for the scaffolding molecule Liprin-α and its interactors Lar and DSyd-1 (RhoGAP100F) in synaptic vesicle recruitment. This suggests that these important synapse regulators might serve a shared role at presynaptic sites within the CNS. In principle, our CLEM approach is broadly applicable to the developmental and ultrastructural analysis of any cell type that can be targeted with genetically encoded markers.

Programmed cell death controlled by ANAC033/SOMBRERO determines root cap organ size in Arabidopsis.

BACKGROUND: The root cap is a plant organ that ensheathes the meristematic stem cells at the root tip. Unlike other plant organs, the root cap shows a rapid cellular turnover, balancing constant cell generation by specific stem cells with the disposal of differentiated cells at the root cap edge. This cellular turnover is critical for the maintenance of root cap size and its position around the growing root tip, but how this is achieved and controlled in the model plant Arabidopsis thaliana remains subject to contradictory hypotheses. RESULTS: Here, we show that a highly organized cell death program is the final step of lateral root cap differentiation and that preparation for cell death is transcriptionally controlled by ANAC033/SOMBRERO. Precise timing of cell death is critical for the elimination of root cap cells before they fully enter the root elongation zone, which in turn is important in order to allow optimal root growth. Root cap cell death is followed by a rapid cell-autonomous corpse clearance and DNA fragmentation dependent on the S1-P1 type nuclease BFN1. CONCLUSIONS: Based on these results, we propose a novel concept in plant development that recognizes programmed cell death as a mechanism for maintaining organ size and tissue homeostasis in the Arabidopsis root cap.

The protein quality control system manages plant defence compound synthesis.

Jasmonates are ubiquitous oxylipin-derived phytohormones that are essential in the regulation of many development, growth and defence processes. Across the plant kingdom, jasmonates act as elicitors of the production of bioactive secondary metabolites that serve in defence against attackers. Knowledge of the conserved jasmonate perception and early signalling machineries is increasing, but the downstream mechanisms that regulate defence metabolism remain largely unknown. Here we show that, in the legume Medicago truncatula, jasmonate recruits the endoplasmic-reticulum-associated degradation (ERAD) quality control system to manage the production of triterpene saponins, widespread bioactive compounds that share a biogenic origin with sterols. An ERAD-type RING membrane-anchor E3 ubiquitin ligase is co-expressed with saponin synthesis enzymes to control the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the rate-limiting enzyme in the supply of the ubiquitous terpene precursor isopentenyl diphosphate. Thus, unrestrained bioactive saponin accumulation is prevented and plant development and integrity secured. This control apparatus is equivalent to the ERAD system that regulates sterol synthesis in yeasts and mammals but that uses distinct E3 ubiquitin ligases, of the HMGR degradation 1 (HRD1) type, to direct destruction of HMGR. Hence, the general principles for the management of sterol and triterpene saponin biosynthesis are conserved across eukaryotes but can be controlled by divergent regulatory cues.

Escherichia coli induces bovine neutrophil cell death independent from caspase-3/-7/-1, but with phosphatidylserine exposure prior to membrane rupture.

Neutrophils are essential for the innate immune response against bacterial pathogens and play a key role during the early phases of infection, including mastitis and endometritis in cows. When directly challenged with bacteria, neutrophils undergo phagocytosis induced cell death (PICD). The molecular mechanisms of this cell death modality are poorly understood, especially for bovine neutrophils. Therefore, this study aimed to determine the mechanisms and hallmarks of PICD in bovine neutrophils after in vitro challenge with Escherichia coli (E. coli). Our data show that various apoptotic hallmarks such as blebbing, chromatin condensation and executioner caspase (C)-3/-7 activity are only observed during constitutive bovine neutrophil apoptosis. In contrast, bovine neutrophil PICD is characterized by production of reactive oxygen species (ROS), pro-inflammatory C-1 activation, nuclear factor (NF)-κB activation, and interleukin (IL)-1ß and IL-6 secretion. Nevertheless, under both conditions these phagocytes undergo cell death with the exposure of phosphatidylserine (PS). Although PS exposure is generally attributed to the anti-inflammatory features of executioner caspase-dependent apoptosis, it surprisingly preceded plasma membrane rupture during bovine neutrophil PICD. Moreover, C-1 inhibition strongly affected IL-1ß production but not the PICD kinetics. This indicates that the secretion of the latter pro-inflammatory cytokine is a bystander effect rather than a regulator of PICD in bovine neutrophils, in marked contrast to the IL-1ß-dependent pyroptosis reported for macrophages.

Colocalization analysis in fluorescence micrographs: verification of a more accurate calculation of pearson's correlation coefficient.

One of the most routine uses of fluorescence microscopy is colocalization, i.e., the demonstration of a relationship between pairs of biological molecules. Frequently this is presented simplistically by the use of overlays of red and green images, with areas of yellow indicating colocalization of the molecules. Colocalization data are rarely quantified and can be misleading. Our results from both synthetic and biological datasets demonstrate that the generation of Pearson's correlation coefficient between pairs of images can overestimate positive correlation and fail to demonstrate negative correlation. We have demonstrated that the calculation of a thresholded Pearson's correlation coefficient using only intensity values over a determined threshold in both channels produces numerical values that more accurately describe both synthetic datasets and biological examples. Its use will bring clarity and accuracy to colocalization studies using fluorescent microscopy.

Alternatively activated macrophages engage in homotypic and heterotypic interactions through IL-4 and polyamine-induced E-cadherin/catenin complexes.

Alternatively activated macrophages (AAMs), triggered by interleukin-4 (IL-4) and IL-13, play a modulating role during Th2 cytokine-driven pathologies, but their molecular armament remains poorly characterized. Here, we established E-cadherin (Cdh1) as a selective marker for IL-4/IL-13-exposed mouse and human macrophages, which is STAT6-dependently induced during polarized Th2 responses associated with Taenia crassiceps helminth infections or allergic airway inflammation. The IL-4-dependent, arginase-1/ornithine decarboxylase-mediated production of polyamines is important for maximal Cdh1 induction, unveiling a novel mechanism for IL-4-dependent gene transcription. At the macrophage surface, E-cadherin forms a functional complex with the catenins that accumulates at sites of cell contact. Macrophage-specific deletion of the Cdh1 gene illustrates the implication of E-cadherin in IL-4-driven macrophage fusion and heterotypic interactions with CD103(+) and KLRG1(+) T cells. This study identifies the E-cadherin/catenin complex as a discriminative, partly polyamine-regulated feature of IL-4/IL-13-exposed alternatively activated macrophages that contributes to homotypic and heterotypic cellular interactions.

Lineage tracing of cardiac explant derived cells.

AIMS: Cultured cardiac explants produce a heterogeneous population of cells including a distinctive population of refractile cells described here as small round cardiac explant derived cells (EDCs). The aim of this study was to explore the source, morphology and cardiogenic potential of EDCs. METHODS: Transgenic MLC2v-Cre/ZEG, and actin-eGFP mice were used for lineage-tracing of EDCs in vitro and in vivo. C57B16 mice were used as cell transplant recipients of EDCs from transgenic hearts, as well as for the general characterisation of EDCs. The activation of cardiac-specific markers were analysed by: immunohistochemistry with bright field and immunofluorescent microscopy, electron microscopy, PCR and RT-PCR. Functional engraftment of transplanted cells was further investigated with calcium transient studies. RESULTS: Production of EDCs was highly dependent on the retention of blood-derived cells or factors in the cultured explants. These cells shared some characteristics of cardiac myocytes in vitro and survived engraftment in the adult heart in vivo. However, EDCs failed to differentiate into functional cardiac myocytes in vivo as demonstrated by the absence of stimulation-evoked intracellular calcium transients following transplantation into the peri-infarct zone. CONCLUSIONS: This study highlights that positive identification based upon one parameter alone such as morphology or immunofluorescene is not adequate to identify the source, fate and function of adult cardiac explant derived cells.

Streptococcus pneumoniae deficient in pneumolysin or autolysin has reduced virulence in meningitis.

BACKGROUND: The role played by pneumolysin and autolysin in pneumococcal meningitis is poorly understood. METHODS: A rat model was used to investigate the disease, in which surgical implantation of a cisternal catheter allowed bacterial instillation and cerebrospinal fluid (CSF) sampling. RESULTS: CSF infection of rats with wild-type pneumococci caused meningitis within 26 h, whereas isogenic mutants that do not express pneumolysin (DeltaPly) or autolysin (LytA(-)) caused very mild or no disease. Wild-type infections resulted in pneumococci in the CSF and cortical homogenates, but a minority of the rats infected with DeltaPly or LytA(-) had bacteria in these locations at 26 h. Leukocyte numbers in the CSF were similar after infection with all pneumococci; however, neutrophils and monocytes predominated after wild-type infection, whereas lymphocytes and atypical lymphocytes predominated after infection with the mutants. Wild-type pneumococci caused disruption to the ependyma, but this was not observed in rats infected with DeltaPly or LytA(-). Cells surrounding the ventricles in wild type-infected animals expressed caspase 3, and astrocytes had hypertrophy; both findings were absent in rats infected with the mutants. CONCLUSIONS: This study provides strong in vivo evidence that pneumolysin and autolysin play crucial roles in the pathogenesis of pneumococcal meningitis.

Neurite degeneration induced by heme deficiency mediated via inhibition of NMDA receptor-dependent extracellular signal-regulated kinase 1/2 activation.

The early stages of many neurodegenerative diseases and age-related degeneration are characterized by neurite damage and compromised synaptic function that precede neuronal cell death. We investigated the signaling mechanisms underlying neurite degeneration using cortical neuron cultures. Inhibition of heme synthesis caused neurite damage, without neuronal death, and was mediated by reduced NMDA receptor (NMDAR) expression and phosphorylation. The signaling toward the degenerative phenotype involved suppression of the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway, and electrophysiological recording showed that the neurodegeneration is accompanied by reduced NMDAR current and Ca2+ influx, as well as reduced voltage-gated sodium currents, consistent with compromised neurite integrity. Rescue from the degenerative phenotype by heme replacement was dependent on restoration of NR2B subunit phosphorylation and expression of NMDAR currents with higher Ca2+ permeability, consistent with triggering prosurvival ERK1/2 signaling to maintain and extend neurites. This study demonstrated a new mechanism of neurodegeneration in which impaired heme synthesis led to NMDAR signaling dysfunction, suppression of the prosurvival ERK1/2 pathway, and progressive fragmentation of neuronal projections.

IgG entry and deposition are components of the neuroimmune response in Batten disease.

Patients and a mouse model of Batten disease, the juvenile form of neuronal ceroid lipofuscinosis (JNCL), raise autoantibodies against GAD65 and other brain-directed antigens. Here we investigate the adaptive component of the neuroimmune response. Cln3(-/-) mice have autoantibodies to GAD65 in their cerebrospinal fluid and elevated levels of brain bound immunoglobulin G (IgG). IgG deposition was found within human JNCL autopsy material, a feature that became more evident with increased age in Cln3(-/-) mice. The lymphocyte infiltration present in human and murine JNCL occurred late in disease progression, and was not capable of central/intrathecal IgG production. In contrast, we found evidence for an early systemic immune dysregulation in Cln3(-/-) mice. In addition evidence for a size-selective breach in the blood-brain barrier integrity in these mice suggests that systemically produced autoantibodies can access the JNCL central nervous system and contribute to a progressive inflammatory response.

Quantization of widefield fluorescence images using structured illumination and image analysis software.

It is difficult to obtain precise quantitative measurements from fluorescent images captured from widefield microscopes. We wished to ascertain if reliable quantitative measurements of both biological and nonbiological specimens were possible using a widefield microscope equipped with a structured illumination system and image analysis software. In a nonbiological specimen, images were obtained from fluorescent beads of known intensity. For a biologically relevant model we used the uptake of fluorescently labeled transferrin by HeLa cells in culture. In the bead sample, the mean intensity of beads acquired with the structured illumination system showed relative intensities near to the predicted values without any further manipulation of data. In the transferrin uptake experiments, uptake and subsequent recycling of the labeled transferrin could be accurately and reproducibly measured. We conclude that using a combination of structured illumination and image processing algorithms accurate quantization of fluorescent images can be achieved in widefield microscopy.

Cleavage of the plasma membrane Na+/Ca2+ exchanger in excitotoxicity.

In brain ischemia, gating of postsynaptic glutamate receptors and other membrane channels triggers intracellular Ca2+ overload and cell death. In excitotoxic settings, the initial Ca2+ influx through glutamate receptors is followed by a second uncontrolled Ca2+ increase that leads to neuronal demise. Here we report that the major plasma membrane Ca2+ extruding system, the Na+/Ca2+ exchanger (NCX), is cleaved during brain ischemia and in neurons undergoing excitotoxicity. Inhibition of Ca2+-activated proteases (calpains) by overexpressing their endogenous inhibitor protein, calpastatin or the expression of an NCX isoform not cleaved by calpains, prevented Ca2+ overload and rescued neurons from excitotoxic death. Conversely, down-regulation of NCX by siRNA compromised neuronal Ca2+ handling, transforming the Ca2+ transient elicited by non-excitotoxic glutamate concentrations into a lethal Ca2+overload. Thus, proteolytic inactivation of NCX-driven neuronal Ca2+ extrusion is responsible for the delayed excitotoxic Ca2+ deregulation and neuronal death.

Focal astrocyte loss is followed by microvascular damage, with subsequent repair of the blood-brain barrier in the apparent absence of direct astrocytic contact.

Blood-brain barrier (BBB) breakdown is a feature of cerebral ischaemia, multiple sclerosis, and other neurodegenerative diseases, yet the relationship between astrocytes and the BBB integrity remains unclear. We present a simple in vivo model in which primary astrocyte loss is followed by microvascular damage, using the metabolic toxin 3-chloropropanediol (S-alpha-chlorohydrin). This model is uncomplicated by trauma, ischaemia, or primary immune involvement, permitting the study of the role of astrocytes in vascular endothelium integrity, maintenance of the BBB, and neuronal function. Male Fisher F344 rats given 3-chloropropanediol show astrocytic damage and death at 4-24 h in symmetrical brainstem and midbrain nuclear lesions, while neurons show morphological changes at 24-48 h. Fluorescent 10 kDa dextran tracers show the BBB leaking from 24 h, progressing to petechial haemorrhage after 48-72 h, with apparent repair after 6 days. BBB breakdown, but not the earlier astrocytic death, is accompanied by a delayed increase in blood flow in the inferior colliculus. An ED1 inflammatory response develops well after astrocyte loss, suggesting that inflammation may not be a factor in starting BBB breakdown. This model demonstrates that the BBB can self-repair despite the apparent absence of direct astrocytic-endothelial contact. The temporal separation of pathological events allows pharmacological intervention, and the mild reversible ataxia permits long-term survival studies of repair mechanisms.