An Economical, Portable Manual Cryogenic Plunge Freezer for the Preparation of Vitrified Biological Samples for Cryogenic Electron Microscopy.

Visualizing biological structures and cellular processes in their native state is a major goal of many scientific laboratories. In the past 20 years, the technique of preserving samples by vitrification has greatly expanded, specifically for use in cryogenic electron microscopy (cryo-EM). Here, we report on improvements in the design and use of a portable manual cryogenic plunge freezer that is intended for use in laboratories that are not equipped for the cryopreservation of samples. The construction of the instrument is economical, can be produced by a local machine shop without specialized equipment, and lowers the entry barriers for newcomers with a reliable alternative to costly commercial equipment. The improved design allows for successful freezing of isolated proteins for single particle analysis as well as bacterial cells for cryo-electron tomography. With this instrument, groups will be able to prepare vitreous samples whenever and wherever necessary, which can then be imaged at local or national cryo-EM facilities.

Coexistence of Fabry disease with IgM nephropathy: A case report.

RATIONALE: Coexistence of Fabry disease and IgM nephropathy is rare. The varying severity and unapparent clinical manifestation of Fabry disease makes it difficult to recognize when coexisting with another more prevalent cause of nephropathy requiring electron microscopy and genetic testing to confirm their coexistence. PATIENT CONCERNS: A 54-year-old female presented with proteinuria without any clinical signs or family history of Fabry disease. DIAGNOSES: Immunostaining of the renal biopsy identified mesangial IgM deposition diagnosing it as IgM nephropathy. The light microscopy indicated prominent vacuolization of podocytes. Further examination of toluidine blue stained semi-thin sections and electron microscopy revealed blue bodies and myelin bodies in the cytoplasm of podocytes, respectively. Mutation analysis detected missense mutation establishing the diagnosis of coexisting Fabry disease. INTERVENTIONS: The patient was treated with angiotensin-converting enzyme inhibitors. Enzyme replacement therapy was not administered due to financial constraints. OUTCOMES: After 2 months of treatment the patient demonstrated urine protein to creatinine ratio of 0.21 g/g. LESSONS: Identifying coexistence of Fabry disease with other nephropathy requires meticulous pathologic investigations including electron microscopy especially when Fabry disease presents with atypical phenotype.

Cell-based two-dimensional morphological assessment system to predict cancer drug-induced cardiotoxicity using human induced pluripotent stem cell-derived cardiomyocytes.

Recent developments of novel targeted therapies are contributing to the increased long-term survival of cancer patients; however, drug-induced cardiotoxicity induced by cancer drugs remains a serious problem in clinical settings. Nevertheless, there are few in vitro cell-based assays available to predict this toxicity, especially from the aspect of morphology. Here, we developed a simple two-dimensional (2D) morphological assessment system, 2DMA, to predict drug-induced cardiotoxicity in cancer patients using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) with image-based high-content analysis in a high-throughput manner. To assess the effects of drugs on cardiomyocytes, we treated iPSC-CMs with 28 marketed pharmaceuticals and measured two key parameters: number of cell nuclei and sarcomere morphology. Drugs that significantly perturbed these two parameters at concentrations ≤30 times the human Cmax value were regarded as positive in the test. Based on these criteria, the sensitivity and specificity of the 2DMA system were 81% and 100%, respectively. Moreover, the translational predictability of 2DMA was comparable with that of a three-dimensional cardiotoxicity assay. RNA sequencing further revealed that the expression levels of several genes related to sarcomere components decreased following treatment with sunitinib, suggesting that inhibition of the synthesis of proteins that comprise the sarcomere contributes to drug-induced sarcomere disruption. Based on these features, the 2DMA system provides mechanistic insight with high predictability of cancer drug-induced cardiotoxicity in humans, and could thus contribute to the reduction of drug attrition rates at early stages of drug development.

A Comparison Study of Real-Time Ultrasound Elastography and Electron Microscopy for the Assessment of Liver Damage Induced by Brain Death.

The aim of this study was to investigate the specificity and sensitivity of real-time ultrasound elastography (RTE) in the evaluation of liver damage induced by brain death and the correlation with ultrastructural changes in liver tissue. Eleven RTE parameters before brain death and at 0, 3, 6 and 9 h after brain death in 12 miniature pigs were collected and analyzed, and the correlation of these parameters with electron microscopy results was explored. Six of the RTE parameters, namely, mean relative strain value within the region of interest, standard deviation of the relative strain value within the region of interest, area of low strain within the region of interest, complexity of low strain area within the region of interest, skewness and correlation, significantly differed among the time periods. Categorical data were analyzed using the χ2-test. Spearman's correlation analysis was used for evaluating correlations between RTE parameters and electron microscopy results, and the correlation coefficients (r) were calculated. Electron microscopy results revealed that liver damage gradually increased after brain death, with significant differences between 0 and 9 h (χ2 = 14.143, p value = 0.027). In addition, the six aforementioned RTE parameters significantly correlated with electron microscopy results, with the mean relative strain value within the region of interest being the strongest (r = -0.59, p value < 0.001) correlated parameter. RTE could provide preliminary assessment of liver damage induced by brain death, and correlates to ultrastructural changes in liver tissue.

In Vitro Approaches for the Assessment of Serpin Polymerization.

Serpin polymerization is the result of end-to-end ordered aggregation of serpin monomers into linear unbranched chains. This change in molecular state represents the basis of several conformational diseases with pathological gain-of-function and loss-of-function phenotypes, termed serpinopathies. Tools that enable quantification and characterization of polymer formation are therefore important to the study of serpin behavior in this pathophysiological context. Such methods rely on different manifestations of molecular change: polymerization-the generation of molecules with increasing molecular weight-is accompanied by concomitant structural rearrangements in the constituent subunits. Different approaches may be appropriate dependent on whether measurements are made on static samples, such as tissue or cell culture extracts, or in time-resolved experiments, often undertaken using polymers artificially induced under in vitro destabilizing conditions. In the former category, we describe the application of polyacrylamide electrophoresis, Western blot, ELISA, and negative-stain electron microscopy and in the latter category, Förster resonance energy transfer and fluorescence spectroscopy using environment-sensitive probes.

Assessment of cardiac fibrosis: a morphometric method comparison for collagen quantification.

Fibrotic remodeling of the heart is a frequent condition linked to various diseases and cardiac dysfunction. Collagen quantification is an important objective in cardiac fibrosis research; however, a variety of different histological methods are currently used that may differ in accuracy. Here, frequently applied collagen quantification techniques were compared. A porcine model of early stage heart failure with preserved ejection fraction was used as an example. Semiautomated threshold analyses were imprecise, mainly due to inclusion of noncollagen structures or failure to detect certain collagen deposits. In contrast, collagen assessment by automated image analysis and light microscopy (LM)-stereology was more sensitive. Depending on the quantification method, the amount of estimated collagen varied and influenced intergroup comparisons. PicroSirius Red, Masson's trichrome, and Azan staining protocols yielded similar results, whereas the measured collagen area increased with increasing section thickness. Whereas none of the LM-based methods showed significant differences between the groups, electron microscopy (EM)-stereology revealed a significant collagen increase between cardiomyocytes in the experimental group, but not at other localizations. In conclusion, in contrast to the staining protocol, section thickness and the quantification method being used directly influence the estimated collagen content and thus, possibly, intergroup comparisons. EM in combination with stereology is a precise and sensitive method for collagen quantification if certain prerequisites are considered. For subtle fibrotic alterations, consideration of collagen localization may be necessary. Among LM methods, LM-stereology and automated image analysis are appropriate to quantify fibrotic changes, the latter depending on careful control of algorithm and comparable section staining.NEW & NOTEWORTHY Direct comparison of frequently applied histological fibrosis assessment techniques revealed a distinct relation of measured collagen and utilized quantification method as well as section thickness. Besides electron microscopy-stereology, which was precise and sensitive, light microscopy-stereology and automated image analysis proved to be appropriate for collagen quantification. Moreover, consideration of collagen localization might be important in revealing minor fibrotic changes.

Capillary electrophoresis, gas-phase electrophoretic mobility molecular analysis, and electron microscopy: effective tools for quality assessment and basic rhinovirus research.

We describe standard methods for propagation, purification, quality control, and physicochemical characterization of human rhinoviruses, using HRV-A2 as an example. Virus is propagated in HeLa-OHIO cells grown in suspension culture and purified via sucrose density gradient centrifugation. Purity and homogeneity of the preparations are assessed with SDS-polyacrylamide gel electrophoresis (SDS-PAGE), capillary electrophoresis (CE), gas-phase electrophoretic mobility molecular analysis (GEMMA), and electron microscopy (EM). We also briefly describe usage of these methods for the characterization of subviral particles as well as for the analysis of their complexes with antibodies and soluble recombinant receptor mimics.

Development of a simple, cost-effective, semi-correlative light and electron microscopy method to allow the immunoelectron localisation of non-uniformly distributed placental proteins.

Immunoelectron microscopy is wrought with technical limitations that complicate its use. However, advances in correlative light and electron microscopy have recently lead to improvements in this field. We report the development of a semi-correlative approach to investigate the ultrastructural location of an antiphospholipid antibody within the syncytiotrophoblast. This method offers several advantages over existing methodologies, since it preserves antigenicity, shows good immunolabel penetrability and does not require specialized equipment. The use of a pre-embedding screen has also allowed us to target individual placental villi and overcome sampling limitations of the electron microscope. This simple, cost-effective method is likely to find widespread application in placental research.

Direct observation of thermal relaxation in artificial spin ice.

We study the thermal relaxation of artificial spin ice with photoemission electron microscopy, and are able to directly observe how such a system finds its way from an energetically excited state to the ground state. On plotting vertex-type populations as a function of time, we can characterize the relaxation, which occurs in two stages, namely a string and a domain regime. Kinetic Monte Carlo simulations agree well with the temporal evolution of the magnetic state when including disorder, and the experimental results can be explained by considering the effective interaction energy associated with the separation of pairs of vertex excitations.

Phenotypic assessment of male fertility status in transgenic animal models.

This chapter describes the approach to define the cause of male infertility in a genetically modified male mouse. It provides a guide to the establishment of the infertility status and whether it is due to the failure of mating or due to abnormalities of the sperm output, motility, and morphology. Further assessments define the nature of the spermatogenic defects and their severity and are designed to determine the pathogenic mechanisms involved.

Improved flow cytometric assessment reveals distinct microvesicle (cell-derived microparticle) signatures in joint diseases.

INTRODUCTION: Microvesicles (MVs), earlier referred to as microparticles, represent a major type of extracellular vesicles currently considered as novel biomarkers in various clinical settings such as autoimmune disorders. However, the analysis of MVs in body fluids has not been fully standardized yet, and there are numerous pitfalls that hinder the correct assessment of these structures. METHODS: In this study, we analyzed synovial fluid (SF) samples of patients with osteoarthritis (OA), rheumatoid arthritis (RA) and juvenile idiopathic arthritis (JIA). To assess factors that may confound MV detection in joint diseases, we used electron microscopy (EM), Nanoparticle Tracking Analysis (NTA) and mass spectrometry (MS). For flow cytometry, a method commonly used for phenotyping and enumeration of MVs, we combined recent advances in the field, and used a novel approach of differential detergent lysis for the exclusion of MV-mimicking non-vesicular signals. RESULTS: EM and NTA showed that substantial amounts of particles other than MVs were present in SF samples. Beyond known MV-associated proteins, MS analysis also revealed abundant plasma- and immune complex-related proteins in MV preparations. Applying improved flow cytometric analysis, we demonstrate for the first time that CD3(+) and CD8(+) T-cell derived SF MVs are highly elevated in patients with RA compared to OA patients (p=0.027 and p=0.009, respectively, after Bonferroni corrections). In JIA, we identified reduced numbers of B cell-derived MVs (p=0.009, after Bonferroni correction). CONCLUSIONS: Our results suggest that improved flow cytometric assessment of MVs facilitates the detection of previously unrecognized disease-associated vesicular signatures.

Electron microscopy of nanoemulsions: an essential tool for characterisation and stability assessment.

The characterisation of pharmaceutical formulations by microscopic techniques is essential to obtain reliable data about the actual morphology of the system. Since the size range of colloidal drug delivery systems has long ago reached the lower end of the nanometer scale, classical light microscopy has been replaced by electron microscopy techniques which provide sufficient resolution for the visualisation of nano-sized structures. Indeed, the superior resolution and methodological versatility of electron microscopy has rendered this technique an indispensable tool for the analysis of nanoemulsions. Microscopic analysis of these lipid-based drug delivery systems with particle sizes in the lower submicron range provides critical information about the size, shape and internal structure of the emulsion droplets. Moreover, surfactant aggregates such as liposomes or multilamellar structures which remain unnoticed during particle size measurements can be detected in this fashion. This review provides a brief overview about both transmission electron microscopy (TEM) and scanning electron microscopy (SEM) techniques which have been employed to characterise nanoemulsions. Of special interest are sophisticated cryo techniques of sample preparation for both TEM and SEM which deliver high-quality images of nanoemulsions in their natural state. An overview about the instrumentation and sample preparation for all presented methods is given. Important practical aspects, sources of error and common artefacts as well as recent methodological advances are discussed. Selected examples of electron microscopic studies of nanoemulsions are presented to illustrate the potential of this technique to reveal detailed and specific information.

Sharp Ca²âº nanodomains beneath the ribbon promote highly synchronous multivesicular release at hair cell synapses.

Hair cell ribbon synapses exhibit several distinguishing features. Structurally, a dense body, or ribbon, is anchored to the presynaptic membrane and tethers synaptic vesicles; functionally, neurotransmitter release is dominated by large EPSC events produced by seemingly synchronous multivesicular release. However, the specific role of the synaptic ribbon in promoting this form of release remains elusive. Using complete ultrastructural reconstructions and capacitance measurements of bullfrog amphibian papilla hair cells dialyzed with high concentrations of a slow Ca²âº buffer (10 mM EGTA), we found that the number of synaptic vesicles at the base of the ribbon correlated closely to those vesicles that released most rapidly and efficiently, while the rest of the ribbon-tethered vesicles correlated to a second, slower pool of vesicles. Combined with the persistence of multivesicular release in extreme Ca²âº buffering conditions (10 mM BAPTA), our data argue against the Ca²âº-dependent compound fusion of ribbon-tethered vesicles at hair cell synapses. Moreover, during hair cell depolarization, our results suggest that elevated Ca²âº levels enhance vesicle pool replenishment rates. Finally, using Ca²âº diffusion simulations, we propose that the ribbon and its vesicles define a small cytoplasmic volume where Ca²âº buffer is saturated, despite 10 mM BAPTA conditions. This local buffer saturation permits fast and large Ca²âº rises near release sites beneath the synaptic ribbon that can trigger multiquantal EPSCs. We conclude that, by restricting the available presynaptic volume, the ribbon may be creating conditions for the synchronous release of a small cohort of docked vesicles.

Wiring economy and volume exclusion determine neuronal placement in the Drosophila brain.

Wiring economy has successfully explained the individual placement of neurons in simple nervous systems like that of Caenorhabditis elegans [1-3] and the locations of coarser structures like cortical areas in complex vertebrate brains [4]. However, it remains unclear whether wiring economy can explain the placement of individual neurons in brains larger than that of C. elegans. Indeed, given the greater number of neuronal interconnections in larger brains, simply minimizing the length of connections results in unrealistic configurations, with multiple neurons occupying the same position in space. Avoiding such configurations, or volume exclusion, repels neurons from each other, thus counteracting wiring economy. Here we test whether wiring economy together with volume exclusion can explain the placement of neurons in a module of the Drosophila melanogaster brain known as lamina cartridge [5-13]. We used newly developed techniques for semiautomated reconstruction from serial electron microscopy (EM) [14] to obtain the shapes of neurons, the location of synapses, and the resultant synaptic connectivity. We show that wiring length minimization and volume exclusion together can explain the structure of the lamina microcircuit. Therefore, even in brains larger than that of C. elegans, at least for some circuits, optimization can play an important role in individual neuron placement.

Neural process reconstruction from sparse user scribbles.

We present a novel semi-automatic method for segmenting neural processes in large, highly anisotropic EM (electron microscopy) image stacks. Our method takes advantage of sparse scribble annotations provided by the user to guide a 3D variational segmentation model, thereby allowing our method to globally optimally enforce 3D geometric constraints on the segmentation. Moreover, we leverage a novel algorithm for propagating segmentation constraints through the image stack via optimal volumetric pathways, thereby allowing our method to compute highly accurate 3D segmentations from very sparse user input. We evaluate our method by reconstructing 16 neural processes in a 1024 x 1024 x 50 nanometer-scale EM image stack of a mouse hippocampus. We demonstrate that, on average, our method is 68% more accurate than previous state-of-the-art semi-automatic methods.

Electron microscopy techniques to study bacterial adhesion.

Since its introduction 70 years ago electron microscopy has become an invaluable tool for microbiology and the study of bacterial interaction. Technological development over the past decades has enabled researchers to resolve smaller and smaller details in bacterial samples, while new preparation techniques like cryo preparation now allow to investigate bacteria even closer to their natural state. In this chapter we give a brief overview of electron microscopy techniques suitable for the investigation of bacterial adhesion at molecular as well as cellular level and a short outlook on future technologies relevant to the field.

MASDET-A fast and user-friendly multiplatform software for mass determination by dark-field electron microscopy.

Electron microscopy has been used to measure the mass of biological nanoparticles since the early 60s, and is the only way to obtain the mass of large structures or parameters such as the mass-per-length of filaments. The ability of this method to sort heterogeneous samples both in terms of mass and shape promises to make it a key tool for proteomics down to the single cell level. A new multiplatform software package, MASDET, that can be run under MATLAB or as a standalone program is described. Based on a user-friendly graphical interface MASDET streamlines mass evaluation and greatly increases the speed of required optimisation procedures. Importantly, the immediate application of Monte-Carlo simulations to describe multiple scattering is possible, allowing the mass analysis of thicker samples and the generation of mass thickness maps.

Ultrashort electron pulses for diffraction, crystallography and microscopy: theoretical and experimental resolutions.

Pulsed electron beams allow for the direct atomic-scale observation of structures with femtosecond to picosecond temporal resolution in a variety of fields ranging from materials science to chemistry and biology, and from the condensed phase to the gas phase. Motivated by recent developments in ultrafast electron diffraction and imaging techniques, we present here a comprehensive account of the fundamental processes involved in electron pulse propagation, and make comparisons with experimental results. The electron pulse, as an ensemble of charged particles, travels under the influence of the space-charge effect and the spread of the momenta among its electrons. The shape and size, as well as the trajectories of the individual electrons, may be altered. The resulting implications on the spatiotemporal resolution capabilities are discussed both for the N-electron pulse and for single-electron coherent packets introduced for microscopy without space-charge.

Assessment of in vitro binding of isolated pectic domains to cellulose by adsorption isotherms, electron microscopy, and X-ray diffraction methods.

Isolated pectic domains representative of the pectic backbone and the neutral sugar side chains were tested for their ability to interact with cellulose in comparison to the well-known binding of xyloglucan. Pectic side chains displayed a significant in vitro binding capacity to cellulose, whereas pectic backbone domains exhibited only slight adsorption to cellulose microfibrils. To support the binding results, electron microscopy and X-ray diffraction were applied. Celluloses from bacteria and sugar beet cell walls were used as substrates for the precipitation of isolated pectic domains or xyloglucan by acetone vapor diffusion. Pectic side chains grew attached to the cellulose surfaces, whereas pectic backbone domains were observed separately from cellulose microfibrils. Xyloglucan seeded with cellulose provoked a decrease of microfibrils entanglement, but no clear cross-links between neighboring microfibrils were observed. These results led to the elucidation of the pectic domains responsible for binding with cellulose microfibrils.