bluuebunny/crossref_metadata_2025 huggingface dataset:
Potassium Permanganate Staining Allows For The Visualization Of An Outer Surface Component Of Selected Ehrlichiae
Authors: Hayes SF , GMunderloh U , Goodman JL , Kocan KM
Published: July 2020

The Ehrlichia spp. are tick-borne pathogens that infect leucocytes and erythrocytes of human beings and animals (1-4). Three ehrlichial organisms, E. chaffeensis, E. equi, HGE (human granulocytic ehrlichiosis agent, equivalent to E. phagocytophila and closely related to E. equi), and Anaplasma  marginale were studied with respect to their morphology after using a combined fixative/potassium permanganate staining protocol (5). Cell cultures infected with the respective organisms were fixed and treated as follows: Tissues were initially fixed with Karnovsky's fixative [glutaraldehydeparaformaldehyde, 2.5/4.0% respectively], containing 0.1% tannic acid, in a Sorenson's phosphate buffer with 0.1M sucrose. Post fixation with OsO4 or without it occurred in some samples. En bloc staining with 1% aqueous uranyl acetate was always done. Dehydration was in a graded ethanol series followed by embedding in Spurr's™ resin. Sample sections were stained for 30-60 minutes at RT with 1% potassium permanganate. These were then washed for 5-15 seconds with 0.025-0.05% sodium citrate, jet washed with dH20, dried and examined (6).
Visualization Of Uptake Of High Density Lipoprotein By Rat Aortic Endothelial Cells In Vitro
Authors: Chao W T , Yang V C
Published: July 2020

The high concentration of low-density lipoprotein in the plasma is the major risk factor of atherosclerosis. On the other hand, another plasma lipoprotein—high-density lipoprotein (HDL) — is inversely correlated with atherosclerosis. Recent studies have demonstrated that HDL mediates the transport of cholesterol from peripheral tissues to the liver. However there is considerable debate about the mechanisms by which high density lipoprotein removes excess cholesterol from cells. Two different pathway were suggested: (i) a docking receptor promoting cholesterol translocation, or (ii) a receptor mediated intracellular endosomal pathway termed “retroendocytosis”. In the present study, we performed immunofluorescence and electron microscopy to directly visualize the uptake of HDL using cholesterol-load rat aortic endothelial and smooth muscle cells.

Endothelial cells were obtained from rat aorta and cultured in medium under 5% CO2 / 95% air atmosphere. Then confluent monolayers of endothelial cells were incubated in cholesterol for 48 hr. The cells were precooled for 2 hr at 4°C with PBS containing HDL-Dil or HDL-gold (10 nm) at a concentration of 80 j± g protein per ml PBS. Internalization experiments were carried out by incubation at 37°C for 0, 5, 15, and 30 min, followed by three washes with PBS (pH 7.4). Then the cells were processed for fluorescence and transmission electron microscopy.
Subcellular Distribution Of Basic Fibroblast Growth Factor-Like Molecules In Fibroblasts And Extracellular Matrix
Authors: Aktas R G , Kayton R J
Published: July 2020

Basic fibroblast growth factor (bFGF) is thought to play an important role in normal tissue repair and wound healing. It is a potent mitogenic and chemotactic factor for fibroblasts. To some degree bFGF regulates proliferation and extracellular matrix (ECM) production by these cells. In this study we investigate (i) the distribution of bFGF in fibroblasts and ECM of supporting tissues (ii) the subcellular location of this protein in vivo and, (iii) mechanisms for release of this growth factor from fibroblasts.

Spurr's resin embedded ultrathin sections from adult rat sciatic nerve, adrenal gland, kidney; and from normal human lung and skin were labeled with either polyclonal (F3393-Sigma) or monoclonal antibodies (F6162-Sigma, C3316-ZymoGenetics Inc.) specific for bFGF using a two-step immunogold labeling method. Controls were incubated with either an equivalent volume of blocking solution (omitting the primary antibody) or an irrelevant antibody (Factor VIII, anti-Histamine)(Fig. 4).
A Post-Embedding Colloidal Gold Immunocytochemical Approach To The Study Of Matrix Accumulation In Glomerular Basement Membrane.
Authors: Miller Caroline A , Cosgrove Dominic
Published: July 2020

Alport renal disease pathogenesis is characterized by a progressive irregular thickening, thinning, and splitting of the glomerular basement membrane (GBM), which culminates in a focal and segmental glomerulonephritis and progressive loss of glomerular filtration, leading to uremia and death. A mouse model for this disease was produced using a gene targeting approach (Cosgrove et al., 1996). The resulting model displays renal pathology that is very similar to that observed in humans. As matrix accumulation has long been associated with the thickened regions of the GBM, this model provided a means to study the molecular composition and ultrastructural localization of matrix in these rarefied regions of the GBM in the Alport mouse.

We examined three matrix molecules based on preliminary data; type IV collagen α l and α2 chains, laminin-1 and fibronectin. Immunohistochemical analysis showed that while all three of these molecules localize primarily to the mesangial matrix of normal mouse glomeruli, in the Alport glomeruli these molecules seem to be heavily deposited in the GBM.
The Primary Cilium of MDCK Cells is a Flow Sensor
Authors: Praetorius H A , Spring K R
Published: July 2020

The primary cilium, a solitary non-motile structure projecting from the centriole of mammalian cells, has been a subject of interest of anatomists since 1898. Surprisingly, the function of the primary cilium in mammalian cells is completely unknown. Virtually all of the epithelial cells of the mammalian kidney, with the exception of the intercalated cells of the collecting duct, express a single primary cilium on their apical (lumenal) surface. These structures were ignored by physiologists until recently when the mechanical properties of the primary cilia of cultured cells of renal origin were measured and it was proposed that they could serve as flow sensors. The proposal stemmed from the observation that flow rates comparable to those observed in renal tubules resulted in a deflection of the cilium of a few microns and that the stiffness of the cilium was commensurate with mechanosensing.

The primary cilia of renal tubular cells are generally about 2 - 3 μm long whereas those of cultured renal cells become much longer as the cells age and may extend 50 μm or more into the medium. MDCK cells, a cultured cell line derived from the collecting duct of the canine kidney, exhibit two cell types analogous to the principal and intercalated cells of the collecting duct. MDCK principal cells express a primary cilium that is visible by high-resolution DIC microscopy. About one week after splitting, the primary cilium of MDCK principal cells is about 8 μm long and is well suited for testing the hypothesis that the cilium serves as a mechanosensor of fluid flow.
Fluorescent Speckle Microscopy in Studies of Cytoskeletal Dynamics During Cell Motility
Authors: Waterman-Storer CM , Salmon W C
Published: July 2020

We have discovered a new method, Fluorescent Speckle Microscopy (FSM), for analyzing the dynamic movement and turnover of macromolecular protein assemblies, such as the cytoskeleton, in living cells (Waterman-Storer et al., 1998). FSM compliments or replaces the techniques of fluorescence recovery after photobleaching or photoactivation of fluorescence for measuring protein dynamics in vivo. For FSM, cells are microinjected with a very low fraction of fluorescently labeled subunits that co-assemble with unlabeled subunits to give a structure with a fluorescent speckled appearance in diffraction-limited wide-field or confocal digital fluorescence images. At low fractions of fluorescent subunits relative to unlabeled subunits, fluorescent speckles vary randomly in intensity according to the number of fluorescent subunits within a diffraction-limited region. in time-lapse FSM image series, movement of the fluorescent speckle pattern indicates translocation of structures, while changes in speckle intensity indicate subunit turnover. We have used FSM to study microtubule and actin behavior in interphase and mitotic cells. We use kymograph analysis to quantitate the movement of speckled structures (Fig 1) and are currently developing analysis procedures to quantify subunit turnover in structures.

We have applied these methods to the study of microtubule and actin cytoskeletal dynamics in migrating vertebrate cells in culture. Interactions between the microtubule and actin cytoskeletons underlie fundamental cellular processes such as cytokinesis and cell locomotion, but are poorly understood.
A spinning disk confocal microscope system for rapid high resolution, multimode, fluorescence speckle microscopy and GFP imaging in living cells
Authors: Maddox Paul , Canman Julie , Grego Sonia , Salmon Wendy , Waterman-Storer Clare , Salmon ED
Published: July 2020

High resolution fluorescent speckle microscopy (FSM) and green fluorescent protein (GFP) imaging in living cells can require image recording at low densities of fluorophores (10 or less/resolvable unit) with low light excitation to prevent photobleaching. This needs efficient optical components, a high quantum efficiency detector, and a digital image acquisition and display system for time-lapse recording of multiple channels. Recently, Shinya and Ted Inoue have described the advantages of the Yokogawa CSU-10 spinning-disk confocal scanning unit for obtaining high quality fluorescent images with brief exposures and low fluorescence bleaching. Based on their findings, we have combined the CSU-10 unit with a high sensitivity pan-chromatic CCD camera to facilitate high spatial and temporal resolution imaging of fluorescence in living cells. in addition, the high signal-to-noise in images obtained with this instrument provides the opportunity to obtain 3-D views of extraordinary resolution and image quality after iterative constrained de-convolution.

Our imaging system is constructed around a Nikon TE300 inverted microscope equipped with either a 60X or 100X Plan Apochromat objective, and standard epi-fluorescence optics for visual inspection of the specimen to locate cells for recording.
Surfing the Conformational Wave of Acetylcholine Receptor Channel Gating
Authors: Cymes Gisela , Grosman Claudio , Auerbach Anthony
Published: July 2020

The muscle nicotinic acetylcholine receptor channel (AChR) is a cylindrical allosteric membrane protein (∼120 x 60 Å Fig. 1) that adopts alternative quaternary conformations (“open” and “closed”) with different functional properties (ion-conducting and ion-impermeable, respectively). We have characterized, residue-by-residue, the dynamics of the conformational change associated with gating using the framework of linear free energy relationships (LFER). The sequence of molecular events that underlies the closed-to-open gating transition was inferred from kinetic measurements of the receptor at the single molecule level.

Specific regions of the AChR were perturbed using site-directed mutagenesis, changes in the membrane potential, or different agonists. Single-channel currents were recorded from cell-attached patches (Fig. 2). For the gain-of-function mutations, choline was used as the agonist because of its low efficacy. The opening rate constant was determined at a saturating concentration of agonist (for choline, 20 mM) in order to isolate gating from binding steps. to avoid bias introduced by fast channel blockade, the closing rate constant was measured at a low concentration (for choline, 200 μM). The diliganded channel opening (β) and closing (α) rate constants were estimated using the QuB suite of kinetic analysis programs. in general, a plot of the log rate constant vs. log equilibrium constant was linear.
Design of a Microscopy System for Quantitative Spatial and Temporal Analysis of Multicellular Interactions
Authors: Sudar D , Callahan D , Parvin B , Knowles D , Ortiz de Solorzano C , Barcellos Hoff MH
Published: July 2020

The challenge of the post-genomic era is functional genomics, i.e. understanding how the genome is expressed to produce myriad cell phenotypes. A phenotype is the result of selective expression of the genome in response to the microenvironment. to use genomic information to understand the biology of complex organisms, the biological responses and signaling pathways in cells need to be studied in context, i.e. within the proper tissue structure. Nonetheless, most current biology is conducted using cells cultured in monolayers on traditional tissue culture plastic. These non-physiological models impede the ability to predict in vivo responses from model systems. The same cells cultured in 2-dimensions (i.e. monolayers) vs. 3-dimensions (e.g. multicellular tumor spheroids) differ in their responses to external stimuli such as ionizing radiation, viral infection, cytotoxic drugs, and chemotherapeutic agents. Our laboratory has led the way in promoting and developing 3-dimensional cell culture models that more accurately reflect in vivo biology, beginning with the establishment 15 years ago of physiologically functional reconstituted mammary acini in culture.

Quantitation of spatial and temporal concurrent behavior of multiple markers in these 3-dimensional cell cultures is hampered by the currently routine mode of sequential image acquisition, measurement and analysis of specific targets. This precludes the detailed analysis of multi-dimensional, time sequence responses and fails to relate features in novel and meaningful ways that will further our understanding of basic biology. Thus new methodology was needed for high-throughput, dynamic evaluations of large numbers of live multicellular specimens. Rather than using confocal microscopy methods, which interfere with live cell systems due to photo-damage, optical sectioning of the 3-dimensional structures is achieved with structured light illumination using the Wilson grating in an implementation described by Lanni.
Visualization of Membrane Sorting and Fusion in Living Cells using Total Internal Reflection (TIR) and Multicolor Video Microscopy
Authors: Toomre Derek , Keller Patrick , Diaz Elena , White Jamie , Simons Kai
Published: July 2020

Post-Golgi sorting of different classes of newly synthesized proteins and lipids is central to the generation and maintenance of cellular polarity. to directly visualize the dynamics and location of apical/basolateral sorting and trafficking we used fast time-lapse multicolor video microscopy in living cells. Specifically, green fluorescent protein color variants (cyan, CFP; yellow, YFP) of apical cargo (GPI-anchored) and basolateral cargo (vesicular stomatitis virus glycoprotein, VSVG) were generated; see FIG 1. Fast dual color fluorescence video microscopy allowed visualization with high temporal and spatial resolution. Our studies revealed that apical and basolateral cargo progressively segregated into large domains in Golgi/TGN structures, excluded resident proteins, and exited in separate transport containers. These carries remained distinct and did not merge with endocytic structures en route to the plasma membrane. Interestingly, our data suggest that the primary sorting occurs by lateral segregation in the Golgi, prior to budding (FIG 2). Further characterization of morphological differences of apical versus basolateral transport carriers was achieved using a specialized microscopy technique called total internal reflection (TIR) microscopy. with this approach only the bottom of the cell (<100 nm) was illuminated by an exponentially decaying evanescent “wave” of light. A series of images, taken at ∼1 second intervals, shows a bright “flash” of fluorescence when the vesicle fuse with the plasma membrane and the fluorophore diffuses into the plasma membrane (FIG 3).
Self-Interference Fluorescent Emission Microscopy for Biological Imaging
Authors: Swan AK , Tong Y , Moiseev L , Ünlü M S , Goldberg BB
Published: July 2020

We present a new method of fluorescence imaging, which yields an unprecedented nm scale vertical resolution. The method uses the unique spectral signature of the fluorescent emission intensity above a reflecting surface to determine vertical position unambiguously. Emission from several heights could be resolved by deconvoluting the spectrum, so that three dimensional imaging is possible. The related technique of standing wave microscopy uses the spatial intensity variation for increased resolution. Applications of this technique include intracellular imaging and screening for specific bacteria, virus or proteins, where discrimination between raised fluorescently labeled specifically bound markers from non-specific binding is crucial.

The emission spectrum of fluorescent markers is modified in the presence of a reflecting surface. Within the wavelength range of the fluorescent emission, the selfinterference of the emitted photons from the direct and reflected path results in enhanced or suppressed emission (constructive or destructive interference) depending on the height and wavelength.
Application of Correlative Microscopy to Genetic Therapy Research of Cystic Fibrosis and Other Human Diseases
Authors: Moore Kenneth C , Wang Guoshen , McCray Paul , Shao Jian , Moninger Tom , Welsh Michael , Nessler Randy
Published: July 2020

Microscopy is an essential tool for developing strategies to use genetic therapy in the treatment of diseases. Negative staining (fig. 1) and cryo-electron microscopy are critical in understanding the structure of native and hybrid vectors to be used as transfer agents. Enzyme cytochemistry is widely used for the detection of markers that aid in determining the rate of gene transfer to cells and tissues. Immunocytochemistry is necessary to identify new gene products. Light, confocal and electron microscopy are used to evaluate pathogenesis, inflammation and cellular changes. For much of the past decade, we have been working to identify the specific gene and cell structure (chloride channel) that is compromised in Cystic Fibrosis patients. Subsequently, appropriate vectors had to be developed for treatment. A significant amount of time and effort has been expended to identify effective viral and other vectors to be used in the transfer of the normal gene construct.

In order to facilitate experimentation, we have developed a cell culture system utilizing normal and cystic fibrosis airway epithelia. These cultures are maintained at an air interface and the cells differentiate into the same types found in vivo.
Study of Distribution and Transport Events of the GluR1 AMPA Receptor: Combination of Genetically Modified Receptors and Multi-Resolution Microscopy
Authors: Stelljes A , Bushong EA , Martone ME , Wiseman PW , Hood KL , Mayford M , Ellisman MH
Published: July 2020

The formation of memory and the process of learning are believed to be regulated, at least in part, by the expression, distribution, and redistribution of glutamate receptors. The expression of these receptors at synaptic sites has a major impact on the strength of synaptic connections, and the AMPA receptor subunit GluR1 appears to play a key role within this system. Increasing evidence suggests that previously silent synapses become activated through rapid AMPA receptor insertion upon appropriate stimulation, and thus the trafficking of this receptor subunit from cellular stores to the synapse is of prime interest.

We are using a transgenic mouse expressing a GFP-tagged form of GluR1 (GluR1-GFP) in order to study the dynamic changes in GluR1 expression and distribution occurring during brain development and following induction of long-term potentiation (LTP). The fusion protein is transcribed under the control of the CaMKIIα promoter, which restricts the localization to forebrain neurons and is coupled to a tetrepressible system, thus allowing for the control of transcription with doxycycline. The anatomical distribution of GluR1-GFP on the light level is consistent with that of wild-type GluR1 (Fig. 1).

We are combining molecular biology with quantitative 3D image analysis on the light and electron microscopic level. The highly fluorescent polar tracer Alexa Fluor 568 (Molecular Probes Inc.) is being used to fill GluR1-GFP expressing pyramidal neurons in tissue slices fixed with aldehydes
3-D Structure Analysis of Escherichia Coli Type 1 Pili
Authors: Hahn E , Wild P , Hermanns U , Sebbel P , Glockshuber R , Haener M , Taschner N , Müller S , Aebi U
Published: July 2020

Type 1 pili are outer membrane organelles formed by 80% of all E. coli strains. They are up to 2 μm long, rod-like appendages covering the bacterial body like spines on a hedgehog (Fig. 1). Type 1 pili expressed by uropathogenic E. coli strains mediate adhesion to specific receptors of the bladder epithelium. They allow colonization of the cell surface as a prelude to infection. Type 1 pili are composed of a rigid rod and a fibrillar tip (Fig. 2a). The minor subunit FimH mediates adhesion and is located at the fibrillar tip as demonstrated by immunolabeling (Fig. 2b). The pilus rod is built from a structural protein, the major subunit FimA. Brinton analyzed parameters describing the helix of the type 1 pilus rod based on paracrystalline arrays. Accordingly, the data predicted one helical repeat to be composed of 25 subunits in 8 turns with a helical pitch of either 2.32 or 2.4 nm.

Here, the 3-D helical structure of the pilus rod has been determined from isolated negatively stained type 1 pili imaged in the annular dark-field mode by scanning transmission electron microscopy (STEM) (Fig. 3a). The power spectra of unbent rods (Fig. 3b) exhibit the strongest layerline at a spacing of (2.42 ± 0.04 nm)−1 representing the pitch of the putative one-start genetic helix (Fig. 3c). The axial distance to its nearest layerline corresponds to the helical repeat (Fig. 3c). Averaging by a linear superposition, as employed for structural analysis of F18 fimbriae, another type of adhering E. coli appendages, confirmed the helical repeat to be 19.31 ± 0.34 nm. Mass-per-length (MPL) determination of unstained type 1 pili by STEM revealed one helical repeat to be composed of 26 ±1 15.8 kDA FimA molecules. All possible helical selection rules were tested by D(Z,k) filtering. Thereby, any data inconsistent with the employed helical selection rule are excluded (Fig. 3d). Hence, the helical selection rule yielding the most power recovery represents the closest selection rule describing the helix. We found l = 8n + 21m to be the optimum helical selection rule. Fig. 3e represents the filtered image. Accordingly, one helical repeat is made of 8 turns containing 27 FimA monomers occurring with an axial rise of 0.72 ± 0.01 nm. D(Z,k) filtering with the helical selection rule assumed by Brinton resulted in significant loss of structure. Hence, we conclude to have refined the previous indexation. The resulting 3-D structure of the type 1 pilus unveils a 6.8 nm wide right handed helical tube harboring an axial cavity (Fig. 4). The tube wall is formed by a coiled fiber comparable to the metal thread of a spring.
Ultrastructural Studies of Root Swelling in Mutants of Arabidopsis Thaliana
Authors: Naidoo Y , Baskin TI , Naidoo G
Published: July 2020

Growth is “anisotropic” when growth rates in different directions are not equal. Anisotropic elongation is controlled by cortical microtubules and cellulose microfibrils of the cell wall. Distorted anisotropic growth results when there are aberrations in either the cellulose network or microtubule cytoskeleton. in this ultrastructural study, the roots of wild type (control) and mutants of Arabidopsis thaliana (L.) Heynh, ecotype Columbia, were compared to determine the role of microtubule organisation, cellulose synthesis and cytokinesis on root expansion.

Three mutations, obtained by treating seeds of A. thaliana with ethane-methylsulfonate and backcrossing once to wild type after four selfed generations, were isolated and designated as rsw (radially swollen). These phenotypes are all temperature sensitive, growing and appearing as wild type at 19°C but expressing the mutant phenotype at or above 30°C. Segments of intact root tips from the three mutants and wild type were prepared and examined for TEM with a Jeol 1200EX. The overall appearance of rsw seedlings at 19°C is the same as wild type. At 30°C these phenotypes lead to extensive swelling of the root apex. Despite considerable alteration in the morphology of the root apex, the appearance of the root cap and root hairs seems essentially normal. When mutant roots expressing these phenotypes at 30°C are returned to 19°C, a wild type appearance of the root apex and elongation gradually returns.

The primary root of the wild type has a remarkably uniform cellular organisation with regularly arranged dense cytoplasmic cells (Fig. 1). At 19°C, rsw4, rsw6 and rsw7 were indistinguishable from wild type; however, at 30°C, the well-defined anatomy was distorted in all tissues by additional cell walls in unusual positions (Fig. 2).
Drought Induced Structural Changes in Developing Chloroplasts of Jalapeno Pepper (Capsicum annuum)
Authors: Fambrough Kristine , Ghoshroy Soumitra
Published: July 2020

Chloroplasts, present in leaves of higher plants have an extensive, folded network of photosynthetic membranes. These membranes have closely appressed (grana) and non-appressed (stroma) regions and they are responsible for the conversion of solar energy into biochemically useful forms. Light plays an important role for the development of mature chloroplasts from proplastids. However, when seeds germinate in the dark, the proplastids do not form mature chloroplasts and instead they form etioplasts. The etioplasts contain a compact, highly regular lattice of inner membranes called prolamellar body. Exposure of dark grown seedlings to light induces formation of fully mature chloroplasts from etioplasts. Plant growth and development is dependent on various environmental factors and drought has profound effect on plant metabolism. Leaves depend on adequate water for both turgor pressure and photosynthesis. Turgor pressure enables nutrients in the form of carbon and minerals to be transported from soil to leaves where they are needed for synthesis of organic compounds. in this study we evaluated the influence of drought on the ultrastructure of developing chloroplasts in pepper plants.

Jalapeno pepper plants were grown from seeds in the dark and drought was induced to experimental group of plants after they developed the first pair of primary leaves. The dark grown control plants received water throughout the drought induction period. At the end of tenth day of drought, plants in both control and experimental groups were exposed to light for up to 48 hours and leaf samples from both groups were obtained at various time intervals after exposure to light. The samples were fixed, dehydrated and embedded for transmission electron microscopy. Ultrathin sections were made, observed under a Hitachi H7000 TEM and data were collected for ultrastructural study. The mature chloroplast and etioplast structures were compared between experimental and control group of plants.
Investigating the Tissue Specific Expression Patterns of Murine Semaphorins 3C, 4D, 4G, 6A and 7A in Primary Lymphoid Tissues
Authors: Chalasani Sreedevi , Matthes David
Published: July 2020

Semaphorins are primarily known for the important role they play in the guidance of growth cones during neuronal development. There is evidence, however, that semaphorins are expressed outside the nervous system as well, suggesting a wider scope for semaphorin function. The overall objective of our study is to identify the functions of semaphorins outside central nervous system especially in T cell development. Some of the 20 semaphorins have been shown to have extra-neural functions that include (for different semaphorins) bone differentiation, promotion of B-cell survival and aggregation, and activation of T-cells. Apart from central nervous system statement of most semaphorins, one semaphorin (CD 100) has transcripts in T cells, B cells, neutrophils, monocytes and granulocytes. EST analysis suggests that other semaphorins are expressed in lymphoid tissues such as thymus, spleen, tonsil, and the interfollicular areas and germinal centers of lymph nodes.

Semaphorins have been related to several cell survival mechanisms, immunosuppression and promotion of cell death resistance. in preliminary studies our lab found that viral semaphorins inhibit the migration of human T cells and human SEMA3A can inhibit migration of human neutrophils.
Three-Dimensional Cellular Organization of Gap Junction Connexins Revealed by Specific Labeling and Electron Tomography
Authors: Sosinsky G , Hand G , Gaietta G , Deerinck T , Martone M , Adams S , Stelljes A , Jordan K , W. Laird D , Tsien R , Ellisman M
Published: July 2020

Gap junction channels serve important functions for the homeostasis of organ systems. These membrane channels serve as low-resistance pathways for the diffusion of small molecular species such as ions, metabolites and second messengers between two neighboring cells (1). Recent intense investigations have been pursuing the effects that defects in gap junction proteins, the connexins, have in hereditary diseases. Mutations in the primary sequence of several connexins have been shown to be responsible for several hereditary human diseases including X-linked Charcot-Marie- Tooth demyelinating neuropathies, nonsyndromic sensorineural deafness, erythrokeratodermia and congenital cataractogenesis (c.f. (2, 3) for recent reviews). Many of the published mutations are being investigated thoroughly to determine at what stage and why the protein fails to make functional channels. Steps involved in connexin trafficking and assembly are: translation of the protein; transport from the endoplasmic reticulum to the Golgi apparatus; oligomerization of the subunits into a hexamer (called a connexon); transport of the connexon to non-junctional areas of the plasma membrane; correct docking with a partner connexon from an apposing cell to form a functional intercellular channel; and incorporation into aggregates (gap junction plaques).
Tomography of Carbon Black for Surface Area Measurement
Authors: Hunt E M , Herd CR
Published: July 2020

Currently, the morphology of carbon black is described by distributive morphological properties that are measured using two dimensional TEM projections of the carbon black aggregates. The area, perimeter and several Feret diameters of 2000 such aggregate projections are measured with an automated system and used to calculate a number of parameters including the mean primary particle diameter and the surface area of the carbon black in m2/g. Several assumptions must be made in order to carry out these calculations, the first being that the aggregates are either isotropic or randomly oriented. True random orientation is impossible due to the flat surface on which the carbon black is deposited. For relatively compact spherical, ellipsoidal and linear aggregates this assumption works fairly well. However, the more highly structured a carbon black aggregate is, the worse this assumption becomes. Studies of these types of aggregates have shown that the two dimensional projected area and perimeter can decrease up to 25% as the viewing angle is increased from 0° to 45°. The use of electron tomography to determine surface area will eliminate this problem. in addition, the use of tomographically reconstructed carbon black aggregates for the determination of surface area is a more direct measurement technique, although limitations to its accuracy do exist.
Novel Molecular Specializations of the Pinceau in Rat Cerebellum as Revealed by Correlated Confocal Microscopy and Electron Tomography
Authors: Bobik Marketta , Capani F , Martone M E , Ellisman M
Published: July 2020

The pinceau is a cerebellar structure formed by GABA-ergic descending basket cell axonal terminals converging on the initial axonal segment of Purkinje cell. Although it exerts a powerful inhibitory influence on the output of the cerebellar cortex, the exact ultrastructural organization and function of this structure are not understood. The unique morphology of the pinceau lies in several elements: lack of synaptic contacts between basket cell terminals and the initial axonal segment, the absence of postsynaptic densities, and heavy ensheathment of the initial segment with glial processes, making astrocytes the only intervening cellular element between the two types of axonal structures. Several plasticity related proteins in this area have been previously characterized by immunocytochemistry. Those include nitric oxide synthase, PSD95, GABA transporters, and several voltage gated potassium channel subunits. We are using a combination of confocal and electron microscopy, correlated photooxidation, EM and tomography to reveal the ultrastructural distribution of novel proteins and their relationships to morphological compartments in the pinceau.

We describe here Kv3.2, a recently discovered member of the Shaw-like subfamily of potassium channels, and the correlated distribution of other proteins described below. Pre-embedding immunolabeling, electron microscopy and tomography showed that Kv3.2 was selectively localized to basket cell axons.
Electron Tomography of Insect Flight Muscle in Isometric Contraction and Rigor States
Authors: Taylor Kenneth A
Published: July 2020

Electron microscopy is a technology fully capable of bridging the light microscopic world with the world of protein structure at atomic resolution. There are numerous examples of cases where cryoelectron microscopy has been used to fit an atomic structure into a structure that has been difficult to crystallize for high resolution structure analysis. More difficult is the fitting of atomic structures into more conventionally prepared tissue like that used in freeze substitution.

We have been using electron tomography to obtain 3-D images of insect flight muscle in various states, including those produced in an active isometric contraction as well as in rigor. These tomograms were obtained using non-uniform tilt angles, cross correlation alignment, area matching and Whittaker-Shannon interpolation of the 3-D transform. The fitting of atomic structures into raw tomograms is a difficult proposition because of the relatively highly noisy feature of the images. Better results are obtained when averages are produced. We have used two method for producing averages. One of these, the so-called column average is used when the muscle filaments are relatively straight with good axial order, or can be straightened in 3-D but their lateral ordering is poor. Column averaging improved considerably reduced the noise in tomograms of contracting muscle enabling the four major crossbridge conformation to be observed (Fig. 1).
Ultrastructure of Bone Mineralization and Osseointegration with Hydroxyapatite-Coated Bone Implants in Vivo
Authors: W. Hobbs Linn , Porter AE , Benezra VI , Blunn GW , Coathup MJ , Spector M
Published: July 2020

The need to optimize adhesion between the femoral stem and surrounding tissue has led to the development of plasma-sprayed HA (PSHA) coatings as attachment facilitators for hip prostheses. The mechanisms underlying this facilitation are incompletely understood, and the long-term efficacy of HA coatings in maintaining bonding to bone remains largely unexplored. in this study, two in vivo models—canine and human—were employed for study by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to elucidate the sequence of early bone mineralization and the longer term fate of HA coatings. High-resolution SEM and TEM of whole bone and anorganic bone were employed to establish the structure of mature bone for comparison.

Whole and Anorganic Bone.Anorganic bone derived from bovine trabecular bone was examined in low-voltage field-emission SEM(LV-FESEM) and high-resolution TEM to establish the morphology of the mineral component of bone [1,2]. Trabeculae were seen to comprise oriented fiber bundles (Fig. 1), each fiber comprising an array of oriented apatite platelets (Fig. 2) arranged like a sheared stack of dominoes with approximately the 64-nm stagger of collagen hole zones.
ESEM Study of Dental Adhesive Polymerization
Authors: Dusevich VM , Eick JD
Published: July 2020

Dental adhesives are bonded to a wet surface of live tissue, dentin. There are two important stages of surface preparation for adhesive application: 1) Etching the tooth structure creating a layer of collagen network free from mineral, and 2) Applying a primer, in this case a water solution of hydrophilic monomer of 2-hydroxyethylmethacrylate (ScotchBond Multipurpose®, 3M). During the priming stage, the resin monomer should penetrate the fibril network and coat collagen fibrils. Subsequent adhesive resin penetration into the collagen network (hybrid layer) is not uniform, influenced by the presence of residual water and degree of resin polymerization. Dependence of bond strength of dental adhesives on time or intensity of light activation is well known, but no direct microscopic observations of the dynamics of polymerization were performed. This was due to the fact that during specimen dehydration for conventional SEM the polymerization process is still proceeding and residual monomer could be washed out.
Molecular-Scale Studies of Proteins at Model Biomaterial Surfaces by Atomic Force Microscopy
Authors: Siedlecki Christopher A
Published: July 2020

A widely accepted tenet of biomaterials research is that the initial step following contact of a synthetic material with blood is the rapid adsorption of plasma proteins. The composition of this adsorbed protein layer is dependent on a variety of factors, including the surface properties of the implant material and the nature of the adsorbing proteins, and the composition and function of this protein layer is important in the subsequent biological response and ultimately the success or failure of the implanted material. While a great amount of effort has gone into developing structure/function responses for implanted biomaterials, there is still much about the molecular level interactions to be determined. We utilized atomic force microscopy (AFM) to investigate the molecular-level interactions of proteins with model biomaterial substrates. The AFM is unique in that it offers the opportunity to characterize interfacial environments, determine material properties, measure protein/surface interaction forces, and visualize the tertiary structure of adsorbed proteins.
Micro-Patterning of Polymers for High Resolution Microscopy Analysis
Authors: Inglis William , Davies Martyn C , Roberts Clive J , Tendler Saul JB , Williams Philip M
Published: July 2020

Micro-patterned surfaces are of interest in biology and chemistry due to the ability to confine functional sample materials to specific areas. If patterns are visible, either through optical microscopy, or topographically, with scanning probe microscopy, investigating samples in a manner that is cheap and accessible to most laboratories is possible. Examples include micropatterning cells in tissue engineering, and protein micro-array analysis. We have created a micro-patterned surface with tailored optical and topographic properties. These were investigated using the microscopy techniques, confocal microscopy (CM), atomic force microscopy (AFM) and near field scanning optical microscopy (NSOM). AFM and CM were used to investigate different aspects of the micro-pattern. to confirm the properties of the micro-pattern, we show the advantages of NSOM in investigating surfaces with both optical and topographic properties simultaneously. Micro-patterns were fabricated using a soft lithography technique, micro-contact printing, where reagents are ‘stamped’ upon substrates using an elastomeric moulding of a micro-template.
Microbial Reduction of Chromium(VI): In-Situ Environmental Cell Transmission Electron Microscopy Study Using Electron Energy Loss Spectroscopy
Authors: Daulton Tyrone L , Little Brenda J , Lowe Kristine , Jones-Meehan Joanne
Published: July 2020

The geochemistry and toxicity of chromium is controlled by its valence state. Chromium is a redox active 3d transition metal with a wide range (− to +6) of possible oxidation states; however only Cr(III) and Cr(VI) are stable. Hexavalent Cr(VI) can be readily reduced to the trivalent state by Fe2+, S2−, organic compounds, wetland plants, and several species of microorganisms. The associated mechanisms of Cr(VI) reduction are technologically and biologically important because they convert a toxic, mobile element into a less toxic, immobile form.

Reduction of Cr(VI) by the bacterium, Shewanella oneidensis (previously classified Shewanella putrefaciens strain MR-1), was studied by absorption spectrophotometry and in-situ, environmental cell (EC) - transmission electron microscopy (TEM) coupled with electron energy loss spectroscopy (EELS). Shewanella oneidensis (S. oneidensis), a gram-negative, facultative bacterium is capable of respiring aerobically and anaerobically using a variety of compounds, including O2, Fe(III), Mn(IV), NO2  −, NO3  −, SO2, SO3  2−, thiosulfate (S2O3  2−), trimethyamine oxide, fumarate, U(VI), and Cr(VI) as terminal electron acceptors.
Holistic Mapping in an Electron Microprobe
Authors: M. MacRae Colin , Wilson NC , Otsuki M
Published: July 2020

When an electron beam interacts with a solid target a number of interactions occur which produce electrons, x-rays and light. Typically in an electron microprobe analyser (EPMA) both the electron and x-ray signals are collected for analysis and imaging. However, if the EPMA is equipped with an optical spectrometer then all three signals can be collected. Commonly, the optical or CathodoLuminescence (CL) spectrometer is a monochromator type and can only collect a single frequency or small range of frequencies at a time. Simultaneous collection of the complete visible spectrum is not possible. The collection optics associated with the spectrometer often must be moved into place to start collection, this then obscures the other detectors and prevents simultaneous collection. At CSIRO Minerals an optical spectrometer has been integrated into a JEOL 8900R EPMA and allows simultaneous collect of all light, x-rays and electron signals. This form of mapping, termed Holistic Mapping, has significant advantages over traditional mapping in that it removes the need to have a priori knowledge about what the important frequencies are that will provide the solution to the problem at hand.
Characterization of Process Induced Contamination and Residues on Semiconductor Components Via FTIR and Raman Microanalysis
Authors: Gotts H
Published: July 2020

FTIR and Raman microanalysis may be used as a powerful combination to determine the identity, and hence infer the source, of contaminant particles which diminish yields of semiconductor components and devices. The complimentarity of these techniques arises from the underlying spectroscopic selection rules.

Vibrational spectroscopic techniques are commonly used to characterize the molecular structure of bulk organic materials. These bulk materials typically represent purified fractions of components which may be further investigated with various classical instrumental techniques such as Differential Scanning Calorimetry (DSC), Nuclear Magnetic Resonance (NMR) spectroscopy, UV-Vis spectroscopy. However, these classical technique may have limited value for the interrogation of small impure particles or materials of limited quantity(ng.).

Elemental techniques such as Scanning Electron Microscopy coupled to Energy Dispersive Spectroscopy are enhanced by the specificity of FTIR Microprobe Spectroscopy and Raman Microprobe Spectroscopy which are now used in process laboratories to characterize and identify particulate and thin film residues with the intent of device yield enhancement.
Industrial Applications of Near-Infrared Chemical Imaging Microscopy
Authors: Lewis EN , Kidder LH , Haber KS
Published: July 2020

Single point near-infrared (NIR) spectroscopy is used extensively for characterizing raw materials and finished products in a wide variety of industries: polymers, paper, film, pharmaceuticals, paintings and coatings, food and beverages, agricultural products. As advanced industrial materials become more complex, their functionality is often determined by the spatial distribution of their discrete sample constituents. However, conventional single point NIR spectroscopy cannot adequately probe the interrelationship between the spatial distribution of sample components with the physical properties of the sample. to fully characterize these samples, it is necessary to probe simultaneously spatial and chemical heterogeneity and correlate these properties with sample characteristics.

Recently, we have developed a novel NIR imaging spectrometer that can deliver spatially resolved chemical information very rapidly. in contrast to conventional, single point NIR spectrometers, the imaging system uses an infrared focal-plane array (FPA) to collect up to 76,800 complete spectra, one for each pixel on the array, in approximately one minute.
How Quantitative are Measurements in the Stem?
Authors: Silcox John
Published: July 2020

The typical electron micrograph is often rich in detail, sometimes too rich, and it can overwhelm the casual observer. The obvious interpretation may be quickly drawn and the next sample brought under study. Yet the electron interaction with the sample is rarely fully exploited. This may be the reason that electron microscopy may sometimes be dismissed as “just pretty pictures”. While in practice “pretty pictures” are not that easy to record or find, it is still true that the interaction of electrons with the sample is complex and therefore able to give the observer much more understanding than is normally found, provided only that the observer is able to unravel the observations. It is the rapid change in this situation that has been fascinating to watch unfurl during the past decades.

In the Scanning Transmission Electron Microscope (STEM) electrons are focused into a tiny spot that is scanned over a thin film specimen.
Electronic Properties of Carbon Nanotubes via Aloof Experiments Using Transmission-EELS
Authors: Reed Bryan W , Sarikaya Mehme
Published: July 2020

Carbon nanotubes (CNT) have great potential as one-dimensional quantum conductors in future molecular electronic applications. Local electronic property determinations at low energies in CNT have been confined to those made by scanning probe microscopy techniques (SPM). Although SPM measurements provide unprecedented electronic property information, they suffer from substrate coupling and a limited ability to measure structural characteristics of nanotubes. A high-resolution transmission electron microscope (TEM) coupled with an electron energy loss spectrometer (EELS) offers electronic property determination as well as structural (imaging and diffraction) and spectroscopic analysis of truly isolated nanotubes. Here we report measurements of EEL spectra of single-walled CNT, both singly and in bundles. The experiments were performed in a VG HB501 STEM at Cornell University, operating at 100 kV. CNT samples chosen for analysis include isolated single-walled tubes with diameters 1.2-1.3 nm and bundles of ∼3-150 single-walled tubes. Many of the spectra were obtained in the aloof mode, i. e. with the beam not penetrating the material.