Whole-mount Transmission Electron Microscopy Research Articles

Identification of Ca-rich dense granules in human platelets using scanning transmission X-ray microscopy.

Whole-mount (WM) platelet preparation followed by transmission electron microscopy (TEM) observation is the standard method currently used to assess dense granule (DG) deficiency (DGD). However, due to the electron-density-based contrast mechanism in TEM, other granules such as α-granules might cause false DG detection. Here, scanning transmission X-ray microscopy (STXM) was used to identify DGs and minimize false DG detection of human platelets. STXM image stacks of human platelets were collected at the calcium (Ca) L2,3 absorption edge and then converted to optical density maps. Ca distribution maps, obtained by subtracting the optical density maps at the pre-edge region from those at the post-edge region, were used to identify DGs based on the Ca richness. DGs were successfully detected using this STXM method without false detection, based on Ca maps for four human platelets. Spectral analysis of granules in human platelets confirmed that DGs contain a richer Ca content than other granules. The Ca distribution maps facilitated more effective DG identification than TEM which might falsely detect DGs. Correct identification of DGs would be important to assess the status of platelets and DG-related diseases. Therefore, this STXM method is proposed as a promising approach for better DG identification and diagnosis, as a complementary tool to the current WM TEM approach.

Platelet Storage Pool Deficiency: Establishment Of Reference Ranges For Platelet Dense Granule Count By Transmission Electron Microscopy

BackgroundPlatelet dense granule (DG) deficiencies, including Hermansky-Pudlak syndrome, are relatively common and frequently under-diagnosed hereditary platelet hypofunctional disorders. Among various laboratory tests, platelet whole mount (WM) transmission electron microscopy (TEM) is considered the gold standard method for diagnosing DG deficiencies. Nevertheless, platelet DG TEM testing is still largely a research tool and has not been thoroughly validated and standardized as a clinical test. Furthermore, reference ranges (RR) of DG count/platelet (DGC) have not been adequately established. AimOur goals were to validate and standardize platelet WM TEM testing and establish reference ranges for platelet DGC. MethodsBased on previously established methods (Blood, 33:598-606), we first optimized and standardized various pre-analytical, analytical and post-analytical procedures including sample matrix, sample stability, platelet-rich plasma (PRP) preparation, platelet mounting procedure and DG counting criteria. Then whole blood samples in ACD-B tubes were collected from a total of 128 healthy donors (64 males and 64 females). Platelet DGC, percentage of platelets without any DG (empty platelets), platelet count (PC), mean platelet volume (MPV) and immature platelet fraction (IPF) were measured. In addition, platelet aggregometry analyses were performed on citrated PRP samples from the same donors. Standard statistical analyses were employed in this study. ResultsDGC and platelet ultra-structure were stable when whole blood samples were stored at room temperature (RT) for up to 4 days. Reproducible DGC and adequate preservation of platelet ultrastructure depended on an optimized mounting method. The DG counting criteria were developed based on previous publications and guidance from Dr. James G. White. Using the same WM TEM images and DG counting criteria, agreements among different technologists (n=5) improved from 60% to 95%. Finally, DGC reference range study using 128 healthy donor samples (age range 18, 72 years) and scoring 100-200 platelets from each donor showed that the DGC distribution of each donor sample was left-skewed with a median of 15% of empty platelets (range: 3, 40). The median DGC was 2.6 with a range of 1.0—5.1. Reference ranges established at the lower 95th percentile for DGC empty platelets is ≤ 31% (95% CI 28, 34) and for the mid-95th percentiles of DGCs between 1.5 and 4.0. DGCs were weakly associated with MPV and IPF (ρ between 0.22 and 0.28); and inversely associated with the percentage of empty platelets (ρ between -0.26 and -0.25). DGC showed no significant association with PC, platelet maximum aggregation or primary slope of aggregation, nor age or gender. ConclusionWe validated platelet WM TEM procedures and for the first time established an adult reference range for platelet DGC. The results of association analyses implied that immature platelets may have slightly more DGs. Disclosures:No relevant conflicts of interest to declare.

Small-Angle X-Ray Scattering for Imaging of Surface Layers on Intact Bacteria in the Native Environment

Crystalline cell surface layers (S-layers) represent a natural two-dimensional (2D) protein self-assembly system with nanometer-scale periodicity that decorate many prokaryotic cells. Here, we analyze the S-layer on intact bacterial cells of the Gram-positive organism Geobacillus stearothermophilus ATCC 12980 and the Gram-negative organism Aquaspirillum serpens MW5 by small-angle X-ray scattering (SAXS) and relate it to the structure obtained by transmission electron microscopy (TEM) after platinum/carbon shadowing. By measuring the scattering pattern of X rays obtained from a suspension of bacterial cells, integral information on structural elements such as the thickness and lattice parameters of the S-layers on intact, hydrated cells can be obtained nondestructively. In contrast, TEM of whole mounts is used to analyze the S-layer lattice type and parameters as well as the physical structure in a nonaqueous environment and local information on the structure is delivered. Application of SAXS to S-layer research on intact bacteria is a challenging task, as the scattering volume of the generally thin (3- to 30-nm) bacterial S-layers is low in comparison to the scattering volume of the bacterium itself. For enhancement of the scattering contrast of the S-layer in SAXS measurement, either silicification (treatment with tetraethyl orthosilicate) is used, or the difference between SAXS signals from an S-layer-deficient mutant and the corresponding S-layer-carrying bacterium is used for determination of the scattering signal. The good agreement of the SAXS and TEM data shows that S-layers on the bacterial cell surface are remarkably stable.

Adsorption and precipitation of iron from seawater on a marine bacteriophage (PWH3A-P1)

The distribution and chemical form of iron in marine environments is of particular interest to oceanographers because of its effect on primary productivity. The high abundance of viruses in the ocean and their small size suggest that they might serve as nucleation centers for iron adsorption and precipitation in the ocean. Hence, we performed laboratory experiments to quantify the adsorption and precipitation of iron by a marine bacteriophage (PWH3a-P1). Acid–base titrations and zeta potential measurements were performed to determine if the virus interacted with protons, and if so, to determine the type, concentration, and reactivity of functional groups present on the viruses. Iron precipitation experiments were conducted to investigate interactions between iron and the viruses. The titrations and zeta potential measurements were well described by a model considering two types of reactive sites on the viruses, with log K values of −4.44 and −8.18, and a total site concentration of 3.64×10 −18 mol per virus. High-magnification observations of the viruses in the presence of iron by whole-mount transmission electron microscopy showed evidence of both adsorption and mineral nucleation. The experimental results clearly demonstrate the potential of marine bacteriophage to serve as nuclei for iron adsorption and precipitation, and the models developed from the data suggest that viruses might represent a significant reservoir of iron in seawater. However, these experiments utilized greatly simplified chemistry and a limited range of iron-to-virus concentration ratios relative to natural seawater, and so the results of this study do not prove that viruses affect the marine iron cycle, but rather serve to justify continued research into iron–virus interactions, in the laboratory and in the field.

Electron microscopic observations of mouse sperm whole mounts after extraction for nuclear matrix and intermediate filaments.

Nuclear matrix and intermediate filaments (NM-IF) can be isolated by sequential treatment with non-ionic detergent, high salt. and nuclease. Extracted cells are easily observed by unembedded whole-mount transmission electron microscopy. Different somatic cell types have been subjected to this procedure and retained their essential architecture. To our knowledge, this work describes the first application of NM-IF extraction to sperm. After chemical dissection the general appearance of mouse sperm cells was preserved, except for head-from-neck separation in some cases. The cell membrane, acrosome and mitochondria were not present. The nucleus showed no apparent changes and revealed no details excepting pore complexes in the posterior part. Tissue-specific cytoskeletal elements (perforatorium, postacrosomal sheath, capitulum, segmented columns, outer dense fibers, submitochondrial reticulum, annulus, and fibrous sheath) were retained, which permitted a parallel between them and intermediate filaments of somatic cells. Tail microtubules were also relatively well preserved, showing high intrinsic stability. Cell structures could be observed well, with some details in the tail even better visible than in ultrathin sections. Observation of mouse sperm whole mounts after NM-IF extraction not only revealed intermediate filament-like properties of their cytoskeletal elements but also offered an additional viewpoint to sperm ultrastructure.

Cytoskeletal Changes in Cultured Human Glaucoma Trabecular Meshwork Cells

Glucocorticoid treatment of cultured human trabecular meshwork (TM) cells has been shown to reorganize actin microfilaments into cross-linked actin networks (CLANs) and to alter TM cell function. The purpose of the present study is to determine whether similar microfilament structural changes occur in TM cells derived from glaucoma patients. The microfilament structures of nine different TM cell cultures, derived from four glaucoma patients and from five normal subjects, was examined by epifluorescent microscopy and by whole-mount transmission electron microscopy. The cells were cultured in the absence and presence of dexamethasone (DEX) for 7-14 days. The normal TM cell lines and glaucoma cell line TM13 had relatively low levels of cross-linked actin networks in the absence of exogenously added glucocorticoids. In contrast, the glaucoma cell lines TM23, TM36C, and TM48D had high levels of CLANs without the addition of dexamethasone. The addition of 10 M DEX to the culture medium significantly increased CLAN expression in all normal as well as glaucoma TM cell lines. CLAN formation in glaucoma TM cells or in TM cells exposed to glucocorticoids may be a factor contributing to the generation of ocular hypertension.

Association of pigmentary organelles, carotenoid droplets, with endoplasmic reticulum in goldfish xanthophores and swordtail fish erythrophores

AbstractA modified procedure for whole mount transmission electron microscopy (WMTEM) was utilized, for electron microscopes with accelerating voltages of 80 to 100 kV, to view the outer 40% of goldfish (Carassius auratus [Linneaus]) xanthophores and swordtail fish (Xiphophorus hellerii [Heckel]) erythrophores. This viewing region covers most of the lamellae and all of the filopodia and cellular processes. In this region one can observe three of the four physiological states: pigment dispersing, pigment fully dispersed, and pigment aggregating. The pigment in the fourth state, fully aggregated, cannot be viewed because of thickness of the cell body and is not addressed in this report. In goldfish xanthophores and swordtail fish erythrophores, the primary pigmentary organelle, the carotenoid droplet, is not a discrete organelle but is either associated with or attached to an extended tubulovesicular structure. Carotenoid droplet translocations appear to be dependent upon external cues which result in the elongation or retraction of these tubulovesicular structures. Tubulovesicular structures are continous with membranous sacs, which we termed carotenoid bodies. Based upon DiOC6(3) staining for the endoplasmic reticulum, we propose that the tubulovesicular structure and the endoplasmic reticulum are the same organelles. By using inhibitors of microfilaments (cytochalasin B or D) and microtubules (nocodazole), we report results which suggest cytoskeletal involvement in the elongation or retraction of endoplasmic reticular cisternae. Results with the intermediate filament inhibitor, cycloheximide, demonstrated that intermediate filaments were eliminated from the cells. We believe that the results are inconclusive because cycloheximide primarily inhibits protein synthesis and any interpretation must be made with care. © 1993 wiley‐Liss, Inc.

Growth cone advance mediated by fibronectin-associated filopodia is inhibited by a phorbol ester tumor promoter

In serum-supplemented medium, exposure to the tumor promoter 4β-phorbol 12β-myristate 13α-acetate (PMA) increases the proportion of SH-SY5Y neuroblastoma cells with neurites and increases the average neurite length. In the present study, under serum-free conditions, PMA treatment had the opposite effects, i.e., retarded neurite sprouting and partially inhibited neurite elongation. This inhibition in neurite outgrowth was partially antagonized by the addition of serum fibronectin (FN) to the medium or substratum. In the absence of PMA, SH-SY5Y cells grown under serum-free conditions showed extensive neurite outgrowth as well as the capacity to secrete FN into their microenvironment and form FN-containing substratum-attachment sites. Immunogold labeling and whole mount transmission electron microscopy (WMTEM) demonstrated FN-containing contact pads at sites where filopodia attached to the substratum and focal plaques on the underside of growth cone margins. The appearance and abundance of FN-containing contact pads and focal plaques were increased by the addition of exogenous FN to defined medium. Focal plaques appeared in close association with microfilament bundles, and nearly always with bundles that projected into filopodia attached to the substratum by contact pads. A method for immunolabeling FN in the filopodial contact pads of living cultures provided more direct evidence that filopodia and contact pads have a major role in FN-mediated attachment and are central in determining growth cone shape and the rate and direction of advance. In support of this view, we show that PMA treatment retards neurite sprouting, alters growth cone morphology and motility, and eliminates the appearance of microfilament bundles, filopodia, and FN-containing substratum-attachment plaques.

Association of bluetongue virus with the cytoskeleton

Analysis of the distribution of [ 36S]methionine-labeled virus proteins following lysis of bluetongue virus (BTV)-infected cells with nonionic detergents showed that a major proportion of the virus-specific proteins was located in the insoluble nuclear-cytoskeletal fraction. Neither the proportion nor the species of virus protein associated with the cytoskeleton was altered following treatment of infected cells with microtubule or microfilament disrupting drugs (colchicine and cytochalasin B, respectively). Electron microscopic examination of BTV-infected cells revealed cytoplasmic virus-specified tubules, viral inclusion bodies (VIB), and progeny virus particles. Whole-mount transmission electron microscopy of nonionic detergent-extracted cells demonstrated the association of VIB, virus particles, and tubules with the cytoskeleton. The identity of virus particles was confirmed with an immunogold labeling technique using a neutralizing monoclonal antibody to BTV protein VP2. Several lines of evidence indicate that virus particles, VIB, and tubules bind to intermediate filaments in BTV-infected cells. (1) These structures remained cytoskeleton associated in infected cells treated with colchicine or cytochalasin B. (2) Linear arrays of filament-associated virus particles were formed around VIB following colchicine-induced juxtanuclear aggregation of intermediate filaments. (3) Virus particles were associated with filaments approximately 10 nm in diameter. (4) Filaments associated with virus particles reacted with an anti-vimentin monoclonal antibody in an immunogold labeling procedure.

Calcium regulation of the actin-mediated cytoskeletal transformation of sea urchin coelomocytes.

Coelomocytes from several echinoderm species undergo an actin-mediated cytoskeletal transformation once subjected to hypotonic shock. In this study, coelomocytes from the sea urchins Lytechinus variegatus and Arbacia punctulata were induced to "transform" by treatment with greater than 5 microM of the calcium ionophore A23187 in the presence of external Ca++. The dependence of ionophore transformation on external Ca++ and the lack of chlorotetracycline staining indicates that these cells rely on external Ca++ sources. NBD-phallacidin (7-Nitrobenz-2-oxa-1,3-diazole-phallacidin) staining of lysolecithin permeabilized cells and whole-mount transmission electron microscopy (TEM) show that similar reorganizations of the actin cytoskeleton take place during hypotonic shock and ionophore transformation, although actin filament bundling is less apparent in A23187-treated cells. As has been shown with hypotonic shock transformation, the ionophore elicited shape change is inhibited by anticalmodulin drugs. Greater than 10 microM concentrations of W 13 inhibit filopod formation, while this drug's less active structural analogue, W 12, exhibits no effects. W 13 also appears to disrupt actin filament-membrane associations in the cells. Fluorescent localization of calmodulin using a photooxidized derivative of trifluoperazine indicates a general cytoplasmic distribution with some concentration in filopod core bundles. Coelomocyte transformation may be an example of a cellular shape change regulated by Ca++ through the action of calmodulin modulation of actin-membrane interactions.