Whole-cell organelle segmentation in volume electron microscopy.

Cells contain hundreds of organelles and macromolecular assemblies. Obtaining a complete understanding of their intricate organization requires the nanometre-level, three-dimensional reconstruction of whole cells, which is only feasible with robust and scalable automatic methods. Here, to support the development of such methods, we annotated up to 35 different cellular organelle classes-ranging from endoplasmic reticulum to microtubules to ribosomes-in diverse sample volumes from multiple cell types imaged at a near-isotropic resolution of 4 nm per voxel with focused ion beam scanning electron microscopy (FIB-SEM)1. We trained deep learning architectures to segment these structures in 4 nm and 8 nm per voxel FIB-SEM volumes, validated their performance and showed that automatic reconstructions can be used to directly quantify previously inaccessible metrics including spatial interactions between cellular components. We also show that such reconstructions can be used to automatically register light and electron microscopy images for correlative studies. We have created an open data and open-source web repository, 'OpenOrganelle', to share the data, computer code and trained models, which will enable scientists everywhere to query and further improve automatic reconstruction of these datasets.

An open-access volume electron microscopy atlas of whole cells and tissues.

Understanding cellular architecture is essential for understanding biology. Electron microscopy (EM) uniquely visualizes cellular structures with nanometre resolution. However, traditional methods, such as thin-section EM or EM tomography, have limitations in that they visualize only a single slice or a relatively small volume of the cell, respectively. Focused ion beam-scanning electron microscopy (FIB-SEM) has demonstrated the ability to image small volumes of cellular samples with 4-nm isotropic voxels1. Owing to advances in the precision and stability of FIB milling, together with enhanced signal detection and faster SEM scanning, we have increased the volume that can be imaged with 4-nm voxels by two orders of magnitude. Here we present a volume EM atlas at such resolution comprising ten three-dimensional datasets for whole cells and tissues, including cancer cells, immune cells, mouse pancreatic islets and Drosophila neural tissues. These open access data (via OpenOrganelle2) represent the foundation of a field of high-resolution whole-cell volume EM and subsequent analyses, and we invite researchers to explore this atlas and pose questions.

Pyrene Excimer-Based Fluorescent Labeling of Cysteines Brought into Close Proximity by Protein Dynamics: ASEM-Induced Thiol-Ene Click Reaction for High Spatial Resolution CLEM.

Fluorescence microscopy (FM) has revealed vital molecular mechanisms of life. Mainly, molecules labeled by fluorescent probes are imaged. However, the diversity of labeling probes and their functions remain limited. We synthesized a pyrene-based fluorescent probe targeting SH groups, which are important for protein folding and oxidative stress sensing in cells. The labeling achieved employs thiol-ene click reactions between the probes and SH groups and is triggered by irradiation by UV light or an electron beam. When two tagged pyrene groups were close enough to be excited as a dimer (excimer), they showed red-shifted fluorescence; theoretically, the proximity of two SH residues within ~30 Å can thus be monitored. Moreover, correlative light/electron microscopy (CLEM) was achieved using our atmospheric scanning electron microscope (ASEM); radicals formed in liquid by the electron beam caused the thiol-ene click reactions, and excimer fluorescence of the labeled proteins in cells and tissues was visualized by FM. Since the fluorescent labeling is induced by a narrow electron beam, high spatial resolution labeling is expected. The method can be widely applied to biological fields, for example, to study protein dynamics with or without cysteine mutagenesis, and to beam-induced micro-fabrication and the precise post-modification of materials.

Optimizing the reaction temperature to facilitate an efficient osmium maceration procedure.

The osmium maceration method is a powerful technique for observing the three-dimensional ultrastructure of cellular organelles by scanning electron microscopy. In the conventional osmium maceration method, tissues are immersed in a diluted osmium tetroxide solution for several days at 20°C to remove soluble cytosolic proteins from the freeze-cracked surface of cells, and the optimal duration of this process is dependent on the cell type. To improve the efficiency of the osmium maceration procedure, we have examined systematically the relationship between the reaction temperature and time of the osmium maceration procedure. Treatment at temperatures higher than 20°C drastically shortened the time required to remove cytosolic proteins from the freeze-cracked surface of specimens with optimal durations for the osmium maceration of hepatocytes at 30, 40, 50 and 60°C being 30, 15, 5 and 1 h, respectively. Considering the stability and reproducibility of the macerated specimens, we concluded that the most appropriate temperature was 30 to 40°C. This rapid osmium maceration procedure was used successfully to observe the 3D ultrastructure of Purkinje cells in the cerebellum and proximal convoluted tubule cells in the kidney. This simple and reproducible rapid osmium maceration protocol should find wide appeal for the 3D analysis of cellular organelles in various cell types.

Restoring drifted electron microscope volumes using synaptic vesicles at sub-pixel accuracy.

Imaging ultrastructures in cells using Focused Ion Beam Scanning Electron Microscope (FIB-SEM) yields section-by-section images at nano-resolution. Unfortunately, we observe that FIB-SEM often introduces sub-pixel drifts between sections, in the order of 2.5 nm. The accumulation of these drifts significantly skews distance measures and geometric structures, which standard image registration techniques fail to correct. We demonstrate that registration techniques based on mutual information and sum-of-squared-distances significantly underestimate the drift since they are agnostic to image content. For neuronal data at nano-resolution, we discovered that vesicles serve as a statistically simple geometric structure, making them well-suited for estimating the drift with sub-pixel accuracy. Here, we develop a statistical model of vesicle shapes for drift correction, demonstrate its superiority, and provide a self-contained freely available application for estimating and correcting drifted datasets with vesicles.

Magnesium/silicon atomic weight percent ratio standards for the tissue identification of talc by scanning electron microscopy and energy dispersive X-ray analysis.

Talc may lodge in human tissues through various routes, and has been associated with the development of ovarian carcinoma in case control epidemiologic studies. Talc is detected in tissues with scanning electron microscopy and energy dispersive X-ray analysis (SEM/EDS), with expected magnesium (Mg) and silicon (Si) peaks. The theoretical atomic weight % Mg/Si ratio for talc is 0.649, and for diagnostic purposes, a range of ± 5% (~0.617 to 0.681) is often used as a standard. Our goal was to establish empirically the quantitative range for talc identification by SEM/EDS using two source materials: a Johnson's Baby PowderTM (cosmetic-grade) consumer sample, and talc from Sigma-Aldrich (particle-grade material intended for scientific use). We examined 401 Mg-Si particles with SEM/EDS across the two samples, using two different SEM microscopes. Overall, we found a mean Mg/Si atomic weight % ratio of 0.645, with minimal differences between study subsets. The standard deviation was 0.025; (± 1σ = 0.620-0.670). The currently used ± 5% diagnostic range (Mg/Si 0.617-0.681) is thus reasonably close to this study's ± 1σ range, and well within a ± 2 σ confidence interval span (Mg/Si 0.595-0.695). The ± 5% range is thus an appropriately conservative standard whose continued use seems justified.

Processing Techniques for Scanning Electron Microscopy Imaging of Giant Cells from Giant Cell Tumors of Bone.

Giant cell tumor (GCT) of bone is a common benign lesion that causes significant morbidity due to the failure of modern medical and surgical treatment. Surface ultra-structures of giant cells (GCs) may help in distinguishing aggressive tumors from indolent GC lesions. This study aimed to standardize scanning electron microscopic (SEM) imaging of GC from GCT of bone. Fresh GCT collected in Dulbecco's Modified Eagle Medium was washed to remove blood, homogenized, or treated with collagenase to isolate the GCs. Mechanically homogenized and collagenase-digested GCs were imaged on SEM after commonly used drying methodologies such as air-drying, tetramethylsilane (TMS)-drying, freeze-drying, and critical point-drying (CPD) for the optimization of sample processing. The collagenase-treated samples yielded a greater number of isolated GC and showed better surface morphology in comparison to mechanical homogenization. Air-drying was associated with marked cell shrinkage, and freeze-dried samples showed severe cell damage. TMS methodology partially preserved the cell contour and surface structures, although the cell shape was distorted. GC images with optimum surface morphology including membrane folding and microvesicular structures on the surface were observed only in collagenase-treated and critical point-dried samples. Collagenase digestion and critical point/TMS-drying should be performed for optimal SEM imaging of individual GCs.

Choosing the number of images and image position when analyzing the UNC Passive Aerosol Sampler for occupational exposure assessment.

The University of North Carolina passive aerosol sampler (UNC sampler) could be an alternative when measuring occupational dust exposure, but the time required for microscopic imaging of the sampler needs to be reduced to make it more attractive. The aims of this study were to (1) characterize the effect on precision when reducing imaging, in order to shorten analysis time and (2) assess if the position of the images makes a difference. Eighty-eight samplers were deployed in different locations of an open pit mine. Sixty images were captured for each UNC sampler, covering 51% of its collection surface, using scanning electron microscopy. Bootstrapped samples were generated with different image combinations, to assess the within-sampler coefficient of variation (CVws) for different numbers of images. In addition, the particle concentration relative to the distance from the center of the sampler was studied. Reducing the number of images collected from the UNC sampler led to up to 8.3% CVws for 10 images when calculating respirable fraction. As the overall CV has previously been assessed to 36%, the additional contribution becomes minimal, increasing the overall CV to 37%. The mean concentrations of the images were modestly related to distance from the center of the sampler. The CVws changed from 8.26% to 8.13% for 10 images when applying rules for the image collection based on distance. Thus, the benefit of these rules on the precision is small and the images can therefore be chosen at random. In conclusion, reducing the number of images analyzed from 60 to 10, corresponding to a reduction of the imaged sampling area from 51% to 8.5%, results in a negligible loss in precision for respirable fraction dust measurements in occupational environments.

Quantitative x-ray microanalysis of model biological samples in the SEM using remote standards and the XPP analytical model.

It is shown that accurate x-ray microanalysis of frozen-hydrated and dry organic compounds, such as model biological samples, is possible with a silicon drift detector in combination with XPP (exponential model of Pouchou and Pichoir matrix correction) software using 'remote standards'. This type of analysis is also referred to as 'standardless analysis'. Analyses from selected areas or elemental images (maps) were identical. Improvements in x-ray microanalytical hardware and software, together with developments in cryotechniques, have made the quantitative analysis of cryoplaned frozen-hydrated biological samples in the scanning electron microscope a much simpler procedure. The increased effectiveness of pulse pile-up rejection renders the analysis of Na, with ultrathin window detectors, in the presence of very high concentrations of O, from ice, more accurate. The accurate analysis of Ca (2 mmol kg-1 ) in the presence of high concentrations of K is possible. Careful sublimation of surface frost from frozen-hydrated samples resulted in a small increase in analysed elemental concentrations. A more prolonged sublimation from the same resurfaced sample and other similar samples resulted in higher element concentrations.

Scanning electron microscopy of chronically implanted intracortical microelectrode arrays in non-human primates.

OBJECTIVE: Signal attenuation is a major problem facing intracortical sensors for chronic neuroprosthetic applications. Many studies suggest that failure is due to gliosis around the electrode tips, however, mechanical and material causes of failure are often overlooked. The purpose of this study was to investigate the factors contributing to progressive signal decline by using scanning electron microscopy (SEM) to visualize structural changes in chronically implanted arrays and histology to examine the tissue response at corresponding implant sites. APPROACH: We examined eight chronically implanted intracortical microelectrode arrays (MEAs) explanted from non-human primates at times ranging from 37 to 1051 days post-implant. We used SEM, in vivo neural recordings, and histology (GFAP, Iba-1, NeuN). Three MEAs that were never implanted were also imaged as controls. MAIN RESULTS: SEM revealed progressive corrosion of the platinum electrode tips and changes to the underlying silicon. The parylene insulation was prone to cracking and delamination, and in some instances the silicone elastomer also delaminated from the edges of the MEA. Substantial tissue encapsulation was observed and was often seen growing into defects in the platinum and parylene. These material defects became more common as the time in vivo increased. Histology at 37 and 1051 days post-implant showed gliosis, disruption of normal cortical architecture with minimal neuronal loss, and high Iba-1 reactivity, especially within the arachnoid and dura. Electrode tracts were either absent or barely visible in the cortex at 1051 days, but were seen in the fibrotic encapsulation material suggesting that the MEAs were lifted out of the brain. Neural recordings showed a progressive drop in impedance, signal amplitude, and viable channels over time. SIGNIFICANCE: These results provide evidence that signal loss in MEAs is truly multifactorial. Gliosis occurs in the first few months after implantation but does not prevent useful recordings for several years. Progressive meningeal fibrosis encapsulates and lifts MEAs out of the cortex while ongoing foreign body reactions lead to progressive degradation of the materials. Long-term impedance drops are due to the corrosion of platinum, cracking and delamination of parylene, and delamination of silicone elastomer. Oxygen radicals released by cells of the immune system likely mediate the degradation of these materials. Future MEA designs must address these problems through more durable insulation materials, more inert electrode alloys, and pharmacologic suppression of fibroblasts and leukocytes.

Scanning technology selection impacts acceptability and usefulness of image-rich content.

OBJECTIVE: Clinical and research usefulness of articles can depend on image quality. This study addressed whether scans of figures in black and white (B&W), grayscale, or color, or portable document format (PDF) to tagged image file format (TIFF) conversions as provided by interlibrary loan or document delivery were viewed as acceptable or useful by radiologists or pathologists. METHODS: Residency coordinators selected eighteen figures from studies from radiology, clinical pathology, and anatomic pathology journals. With original PDF controls, each figure was prepared in three or four experimental conditions: PDF conversion to TIFF, and scans from print in B&W, grayscale, and color. Twelve independent observers indicated whether they could identify the features and whether the image quality was acceptable. They also ranked all the experimental conditions of each figure in terms of usefulness. RESULTS: Of 982 assessments of 87 anatomic pathology, 83 clinical pathology, and 77 radiology images, 471 (48%) were unidentifiable. Unidentifiability of originals (4%) and conversions (10%) was low. For scans, unidentifiability ranged from 53% for color, to 74% for grayscale, to 97% for B&W. Of 987 responses about acceptability (n=405), 41% were said to be unacceptable, 97% of B&W, 66% of grayscale, 41% of color, and 1% of conversions. Hypothesized order (original, conversion, color, grayscale, B&W) matched 67% of rankings (n=215). CONCLUSIONS: PDF to TIFF conversion provided acceptable content. Color images are rarely useful in grayscale (12%) or B&W (less than 1%). Acceptability of grayscale scans of noncolor originals was 52%. Digital originals are needed for most images. Print images in color or grayscale should be scanned using those modalities.

Soft X-ray spectromicroscopy for speciation, quantitation and nano-eco-toxicology of nanomaterials.

There is a critical need for methods that provide simultaneous detection, identification, quantitation and visualization of nanomaterials at their interface with biological and environmental systems. The approach should allow speciation as well as elemental analysis. Using the intrinsic X-ray absorption properties, soft X-ray scanning transmission X-ray spectromicroscopy (STXM) allows characterization and imaging of a broad range of nanomaterials, including metals, oxides and organic materials, and at the same time is able to provide detailed mapping of biological components. Thus, STXM offers considerable potential for application to research on nanomaterials in biology and the environment. The potential and limitations of STXM in this context are discussed using a range of examples, focusing on the interaction of nanomaterials with microbial cells, biofilms and extracellular polymers. The studies outlined include speciation and mapping of metal-containing nanomaterials (Ti, Ni, Cu) and carbon-based nanomaterials (multiwalled carbon nanotubes, C60 fullerene). The benefits of X-ray fluorescence detection in soft X-ray STXM are illustrated with a study of low levels of Ni in a natural river biofilm.

ATP bioluminescence values are significantly different depending upon material surface properties of the sampling location in hospitals.

BACKGROUND: Our previous study into assessing hospital cleanliness in Japan by two common methods, ATP bioluminescence and the stamp agar method, revealed considerable variability in the data of both methods (BMC Research Notes, 7: 121, 2014). To investigate the reason(s) for the variability, we reanalyzed the data (n = 752) from the point of view of the material surface properties of sampling sites. METHODS: Data obtained from surfaces with unknown properties and different purposes such as floor were omitted, and the remaining data (n = 488) were used for this study. The material surface properties on sampling sites were divided into six categories: melamine coated (n = 216), vinyl chloride (n = 16), stainless steel (n = 144), wood (n = 63), and acrylonitrile-butadiene styrene resin coated (n = 48). The data between individual material properties were compared. RESULTS: The ATP values of high-touch places were significantly different depending on the type of surface, but no significant difference in stamp values between material properties was seen, indicating that in contrast to stamp values, ATP-accumulation more depends on the physical properties of the material surface such as electronic charges or roughness. To confirm this, we assessed a degree of roughness on vinyl chloride material surface (disutilized floor samples actually used for each of the hospitals) by observation with scanning electron microscope (SEM). As a result, SEM observation similarly revealed considerable roughness on the materials, which may allow microbes to contaminate the materials without noticing it. CONCLUSION: Material properties must be considered when evaluating hospital cleanliness with ATP values, and provide a strong warning into evaluating hospital cleanliness.

Assessing the precision of strain measurements using electron backscatter diffraction--part 1: detector assessment.

We analyse the link between precision of pattern shift measurements and the resolution of the measurement of elastic strain and lattice rotation using high resolution electron backscatter diffraction (HR-EBSD). This study combines analysis of high quality experimentally obtained diffraction patterns from single crystal silicon; high quality dynamical simulations using Bloch wave theory; quantitative measurements of the detector Modulation Transfer Function (MTF) and a numerical model. We have found that increases in exposure time, when 1×1 binning is selected, are the primary reason for the observed increase in sensitivity at greater than 2×2 binning and therefore use of software integration and high bit depth images enables a significant increase in strain resolution. This has been confirmed using simulated diffraction patterns which provide evidence that the ultimate theoretical resolution of the cross correlation based EBSD strain measurement technique with a 1000×1000 pixel image could be as low as 4.2×10(-7) in strain based on a shift precision of 0.001 pixels.

A new quantitative method for gunshot residue analysis by ion beam analysis.

Imaging and analyzing gunshot residue (GSR) particles using the scanning electron microscope equipped with an energy dispersive X-ray spectrometer (SEM-EDS) is a standard technique that can provide important forensic evidence, but the discrimination power of this technique is limited due to low sensitivity to trace elements and difficulties in obtaining quantitative results from small particles. A new, faster method using a scanning proton microbeam and Particle Induced X-ray Emission (µ-PIXE), together with Elastic Backscattering Spectrometry (EBS) is presented for the non-destructive, quantitative analysis of the elemental composition of single GSR particles. In this study, the GSR particles were all Pb, Ba, Sb. The precision of the method is assessed. The grouping behaviour of different makes of ammunition is determined using multivariate analysis. The protocol correctly groups the cartridges studied here, with a confidence >99%, irrespective of the firearm or population of particles selected.

Formulation and Evaluation of Sustained Release Tablets of Metformin Hydrochloride by Solid Dispersion Technique Using pH dependent and pH independent Eudragit Polymers

Objectives: The purpose of the present investigation was to evaluate the influence of solid dispersion of pH dependent and pH independent Eudragit polymers on the sustained release metformin hydrochloride matrix tablets. Materials and methods: Matrix formulations were prepared by direct compression techniques. The excipients used in this study did not alter physicochemical properties of the drug, as tested by FTIR and DSC. All the batches were evaluated various physical parameters. The in vitro drug dissolution and SEM studies were also carried out. Mean dissolution time is used to characterize drug release rate from a dosage form. Results and discussion: Among the different examined polymer blends, Eudragit RLPO with S100 and Ll00 matrix tablets based on solid dispersion showed highly sustained release pattern. Kinetic modeling of in vitro dissolution profiles revealed the drug release mechanism ranges from diffusion controlled to anomalous type. Conclusions: Fitting the data to Korsmeyer equation indicated that diffusion along with erosion could be the mechanism of drug release(AU)

Objetivos: El propósito de la presente investigación ha consistido en evaluar la influencia de la dispersión sólida de polímeros pH dependientes y pH independientes de Eudragit en los comprimidos de matriz de liberación prolongada de clorhidrato de metformina. Métodos: Las formulaciones de matriz se han preparado a través de técnicas de compresión directa. Los excipientes utilizados en este estudio no han modificado las propiedades fisicoquímicas del fármaco, tal y como se ha probado mediante FTIR y DSC. Todos los lotes se han evaluado en varios parámetros físicos. También se ha llevado a cabo la disolución del fármaco in vitro y estudios de SEM (Microscopio electrónico de barrido). El tiempo medio de disolución se utiliza para describir el índice de liberación del fármaco de una forma farmacéutica. Resultados y Discusión: Entre las distintas mezclas de polímeros examinadas, los comprimidos de matriz de Eudragit RLPO con S100 y Ll00 basados en una dispersión sólida han mostrado un patrón de liberación muy prolongado. El modelado cinético de los perfiles de la disolución in vitro ha revelado el margen de mecanismo de liberación del fármaco desde una difusión controlada hasta un tipo anómalo. Conclusiones: La adecuación de los datos con la ecuación de Korsmeyer ha indicado que la difusión junto con la erosión podría ser el mecanismo de la liberación del fármaco(AU)

A systematic method for predetermined scanning electron microscope analysis in dental science.

Scanning electron microscope evaluation could be criticized if the method adopted to correct for bias is not specified in the study design. Observers can draw conclusions from images unconsciously chosen to best support their research hypotheses, impairing the basic research principle of operator's impartiality. In this study, a systematic observation method has been described and verified for repeatability. The number and the observation points on a certain specimen have been predetermined using a scheme along with observation rules previously established in the research protocol. When our instrument is used at an operating magnification between 500x and 1,000x (corresponding to a frame of 250x190 micro and 120x90 micro, respectively), the method allowed 100% repeatable observation frames, with linear frame errors in finding an observation point of 12.5% in length and 16.8% in height. With modifications to accommodate research objective and statistical requirements, the method could be applied to many SEM observation study.

Bone changes after maxillary sinus surgery: an experimental scanning electron microscopy study.

AIMS: The purpose of this study was to explore the pathogenic role of bone in sinus surgery, and to determine whether mucosal disease after sinus surgery may be induced by underlying disease within the bone. MATERIAL AND METHODS: Twenty-five rabbits were divided into five groups. In group one, wide surgical removal of the maxillary sinus mucosa and creation of a nasoantral window were undertaken. In group two, only nasoantral window creation was undertaken. In group three, the mucosa of the anterior maxillary sinus was removed. In group four, a strip of mucosa around the ostium was removed. Group five was used as a control. RESULTS: After three months, in the groups which had undergone wide surgical removal, the medial sinus walls were observed to be retracted and the inner curtain of the bone disturbed, with alteration of the haversian canal system and osteoclastic bone resorption. CONCLUSIONS: After radical sinus surgery, electron microscopic changes were observed in the bony walls, similar to those changes seen in osteomyelitis.

Improvement on the visualization of cytoskeletal structures of protozoan parasites using high-resolution field emission scanning electron microscopy (FESEM).

The association of high resolution field emission scanning electron microscopy (FESEM), with a more efficient system of secondary electron (SE) collection and in-lens specimen position, provided a great improvement in the specimen's topographical contrast and in the generation of high-resolution images. In addition, images obtained with the use of the high-resolution backscattered electrons (BSE) detector provided a powerful tool for immunocytochemical analysis of biological material. In this work, we show the contribution of the FESEM to the detailed description of cytoskeletal structures of the protozoan parasites Herpetomonas megaseliae, Trypanosoma brucei and Giardia lamblia. High-resolution images of detergent extracted H. megaseliae and T. brucei showed the profile of the cortical microtubules, also known as sub-pellicular microtubules (SPMT), and protein bridges cross-linking them. Also, it was possible to visualize fine details of the filaments that form the lattice-like structure of the paraflagellar rod (PFR) and its connection with the axoneme. In G. lamblia, it was possible to observe the intricate structure of the adhesive disk, funis (a microtubular array) and other cytoskeletal structures poorly described previously. Since most of the stable cytoskeletal structures of this protozoan rely on tubulin, we used the BSE images to accurately map immunolabeled tubulin in its cytoskeleton. Our results suggest that the observation of detergent extracted parasites using FESEM associated to backscattered analysis of immunolabeled specimens represents a new approach for the study of parasite cytoskeletal elements and their protein associations.