Low-voltage transmission electron microscopy reveals SV40 viral particles within secretory granules in pancreatic cells

Novel approach in low voltage transmission electron microscopy (TEM) has revealed the presence of SV40 viral like particles in the secretory zymogen granules and in spherical membrane-bound dense bodies of SV40 infected pancreatic cells. The presence of SV40 antigen in these cellular compartments was confirmed by immunocytochemistry of the VP1 antigen. Visualization of the viral particles was only possible by examining ultrathin tissue sections with low-voltage TEM that significantly enhances imaging contrast. Results indicate that following infection of the cell entry and trafficking of the viral particles are present in unique cellular compartments such as ER, dense bodies, and secretory granules.     

Differential morphometric values induced in Golgi apparatus of higher plant cells by aldehyde and permanganate fixation

In order to determine the best conditions to carry out quantitative ultrastructural studies in plant specimens, five different fixation techniques, including some of the most reported electron microscopy fixatives (glutaraldehyde-paraformaldehyde, osmium tetroxide, potassium permanganate), were assayed in onion root meristems to check their ability to induce morphometric changes in Golgi apparatus ultrastructure. Although the parameters evaluated showed in all cases the same tendency, values obtained after permanganate fixation were always higher than those found after aldehyde techniques (especially aldehyde-osmium). Aldehyde followed by osmium fixation appears as the most indicated fixation method when accurate quantitative ultrastructural studies are to be developed.     

Methods for additive hydration allowing observation of fully hydrated state of wet samples in environmental SEM

Methods for additive hydration are presented that enable longtime observation of very wet biological specimens in an environmental scanning electron microscope. The changes of structure due to dehydration for specimens placed on a Peltier-cooled holder, put on a special agar base or embedded in it or blown over by water vapor are compared. The degree of dehydration damage of the observed specimen structures is evaluated and compared with the structure of a nondestructively dried specimen, prepared by the critical point drying method.     

Imaging and measurement of cytosolic free calcium in plant and fungal cells

Calcium plays a central role as a second messenger in plant and fungal cells and as such is involved in controlling numerous biological processes. Direct demonstration of signal-response coupling via Ca²⁺ requires the measurement and localization of changes in cytosolic free Ca²⁺, [Ca²⁺]_i, during these processes in living cells. In recent years this has become possible with the introduction of a range of fluorescent dyes (e.g. Indo-1 Fura-2 and Fluo-3) which have a high affinity and selectivity for free Ca²⁺. When used with recently developed microscope technologies (e.g. fluorescence ratio imaging or confocal scanning laser microscopy), subcellular localization and precise quantification of [Ca²⁺]_i dynamics in single cells can be achieved. This review describes the principles of [Ca²⁺]_i imaging and measurement using fluorescent dyes, the equipment required to do it, the problems with botanical material and how they are being overcome, future developments for this approach in plant cell biology, and an entirely different strategy for the imaging and measurement of [Ca²⁺]_i involving genetic transformation with the aequorin gene.     

Imaging intracellular elemental distribution and ion fluxes in cultured cells using ion microscopy: a freeze-fracture methodology

A freeze-fracture methodology was standardized for tissue culture cells to study intracellular distribution of diffusible elements with ion microscopy. Chinese hamster ovary (CHO) and normal rat kidney (NRK) cells grown on a silicon substrate were sandwiched using another smooth surface (silicon, glass, mica) in the presence of spacers and fast frozen in liquid nitrogen slush. The sandwich was fractured by prying the two halves apart under liquid nitrogen. This procedure produced large areas on the silicon substrate containing hundreds of cells grouped together and fractured at the apical cell surface. After freeze-drying, these cells revealed a subcellular distribution of Na, K, Ca, Mg, P, Cl and S with the ～0·5 μm lateral resolution of the ion microscope. Between the nuclei and the cytoplasm of cells, no major differences were observed for Na, K, Mg, P, Cl and S intensities. Calcium alone, however, exhibited a remarkable distribution. Calcium accumulated more in the cytoplasm than in the nuclei of cells. Even within the cytoplasm its distribution was heterogeneous, suggesting Ca binding sites. The fractured cells consistently exhibited high K-low Na intensities. The injured or dead cells were easily recognized among the healthy ones due to their abnormal ion composition. This simple freeze-fracture methodology allowed fracturing of cells without removing the cells from the substrate. In addition, it eliminated the need for washing the nutrient media away and cryo-sectioning before ion microanalysis. The methodology was successfully extended to 3T3 mouse fibroblast, PtK₂ rat kangaroo and L5 rat myoblast cultures.     

Destruction of polymer growth substrates for cell cultures in two-photon microscopy

The choice of the growth substrate for cell cultures used in fluorescence microscopy is guided by several factors including the type of cells studied and the type of microscopy used. Usually, cells can be cultured on either polymer or glass substrates. One type of polymer, termed Aclar, presents several attractive features: the adhesive properties are better than those of glass, the optical properties are comparable to those of glass, it is biochemically inert, unbreakable, flexible and has a high surface tension, convenient for seeding cells on the cover slip. However, here we show that when imaging with two-photon microscopy, which is based on a femtosecond pulsed laser source, local damage of the Aclar substrate occurs, starting at an average intensity of 10⁵ W cm⁻² at the focal point and for exposure times insufficient to cause cell damage. This leads to the appearance of gas bubbles on cultures plated on Aclar cover slips, which perturb the imaging. By contrast, this phenomenon does not occur on borosilicate cover slips, probably because of their different physical (thermal conductivity, absorbance, melting point) and material homogeneity properties. Thus, for cell culture applications using pulsed lasers with high intensities, the use of glass is preferable to Aclar. The results also reveal that substrates can be more susceptible to thermal damage than the cells themselves.     

Three-dimensional distribution of damaged cells in cryopreserved pancreatic islets as determined by laser scanning confocal microscopy

The technique of serial optical sectioning by confocal microscopy, in conjunction with off-line digital image analysis, was used to quantify the radial distribution of damaged cells in rat pancreatic islets following cryopreservation. The process consists of imaging frozen-thawed islets of Langerhans using laser scanning confocal microscopy (LSCM). The three-dimensional (3-D) distribution and analysis of the two populations of viable and damaged cells was visualized via acridine orange/propidium iodide (AO/PI) fluorescent staining. In preparation for cryopreservation, isolated and cultured rat pancreatic islets were brought to a 2 m concentration of dimethyl sulphoxide (DMSO) by serial addition at decreasing temperatures. Ice was nucleated in the islet suspension at ?10°C, and individual specimens were frozen to ?70°C at cooling rates of 1, 3, 10 and 30°C/min in a programmable bulk freezer and subsequently stored in liquid nitrogen. After rapid thawing and serial dilution to remove DMSO, individual islets were prepared with AO/PI stains for imaging on the LSCM. Serial sections of the islets, 2–7 μm in thickness, were obtained and processed to obtain high-contrast images. Analysis algorithms consisted of template masking, grey-level thresholding, median filtering and 3-D blob colouring. The radial distribution of damaged cells in the islets was determined by isolating the cell and computing its distance from the centroid of the 3-D islet volume. An increase in the number of blobs corresponding to single and/or aggregates of damaged cells was observed progressively with distance from the centre towards the periphery of the islet. This pattern of freeze-induced killing of cells within the islet was found to occur consistently in the numerous individual specimens processed.     

A method for studying the three-dimensional organization of cytoskeletal elements of cells: improvements in the polyethylene glycol technique

A method utilizing polyethylene glycol (PEG) as an extractable embedment for electron microscopy is described. Tissues are fixed according to conventional protocols, embedded in PEG, and sectioned. Sections (ranging from 100 to 500 nm in thickness) are mounted on grids, divested of their PEG matrix, critical-point-dried, and examined stereoscopically. This method greatly facilitates studies on the three-dimensional organization of cytoskeletal and cytoplasmic contractile systems in both muscle and nonmuscle cells.     

Evaluation of fracture planes and cell morphology in complementary fractures of cultured cells in the frozen-hydrated state by field-emission secondary electron microscopy: feasibility for ion localization and fluorescence imaging studies

We have employed field-emission secondary electron microscopy (FESEM) for morphological evaluation of freeze-fractured frozen-hydrated renal epithelial LLC-PK₁ cells prepared with our simple cryogenic sandwich-fracture method that does not require any high-vacuum freeze-fracture instrumentation (Chandra et al. (1986) J. Microsc. 144 , 15–37). The cells fractured on the substrate side of the sandwich were matched one-to-one with their corresponding complementary fractured faces on the other side of the sandwich. The FESEM analysis of the frozen-hydrated cells revealed three types of fracture: (i) apical membrane fracture that produces groups of cells together on the substrate fractured at the ectoplasmic face of the plasma membrane; (ii) basal membrane fracture that produces basal plasma membrane-halves on the substrate; and (iii) cross-fracture that passes randomly through the cells. The ectoplasmic face (E-face) and protoplasmic face (P-face) of the membrane were recognized based on the density of intramembranous particles. Feasibility of fractured cells was shown for intracellular ion localization with ion microscopy, and fluorescence imaging with laser scanning confocal microscopy. Ion microscopy imaging of freeze-dried cells fractured at the apical membrane revealed well-preserved intracellular ionic composition of even the most diffusible ions (total concentrations of K⁺, Na⁺ and Ca⁺). Structurally damaged cells revealed lower K⁺ and higher Na⁺ and Ca⁺ contents than in well-preserved cells. Frozen-freeze-dried cells also allowed imaging of fluorescently labelled mitochondria with a laser scanning confocal microscope. Since these cells are prepared without washing away the nutrient medium or using any chemical pretreatment to affect their native chemical and structural makeup, the characterization of fracture faces introduces ideal sample types for chemical and morphological studies with ion and electron microscopes and other techniques such as laser scanning confocal microscopy, atomic force microscopy and near-field scanning optical microscopy.     

Electron microscopy of melamine-embedded frog retina: evidence for the overall crystalline organization of photoreceptor outer segments

Melamine-formaldehyde, a widely used technical aminoplast, was recently introduced as a water-soluble embedding-medium for electron microscopy (Bachhuber & Frösch, 1983). When processed, using this new technique, the membranes and associated cytoplasmic structures of rod and cone discs show a high degree of structural organization. The membranes are symmetrical and composed of arrays of a 2–2·5 nm particle with a very regular spacing of 3 nm. A network of delicate filaments of less than 2 nm is superimposed on to this framework of membranes, running perpendicularly between the discs and across the disc membranes. The outer segment as a whole shows a crystal-like organization.     

Fluorescence localization after photobleaching (FLAP): a new method for studying protein dynamics in living cells

FLAP is a new method for localized photo‐labelling and subsequent tracking of specific molecules within living cells. It is simple in principle, easy to implement and has a wide potential application. The molecule to be located carries two fluorophores: one to be photobleached and the other to act as a reference label. Unlike the related methods of fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP), the use of a reference fluorophore permits the distribution of the photo‐labelled molecules themselves to be tracked by simple image differencing. In effect, FLAP is therefore comparable with methods of photoactivation. Its chief advantage over the method of caged fluorescent probes is that it can be used to track chimaeric fluorescent proteins directly expressed by the cells. Although methods are being developed to track fluorescent proteins by direct photoactivation, these still have serious drawbacks. In order to demonstrate FLAP, we have used nuclear microinjection of cDNA fusion constructs of β‐actin with yellow (YFP) and cyan (CFP) fluorescent proteins to follow both the fast relocation dynamics of monomeric (globular) G‐actin and the much slower dynamics of filamentous F‐actin simultaneously in living cells.     

空间连杆机构中四面体法的单元形成研究

本文探讨了空间连杆机构中四面体法的单元形成原则与方法,提出了“虚单元”概念和坐标系固化的策略,并以实际机构为例具体阐述了四面体法解决问题的包含单元形成和单元求解的完整的步骤与方法,较好地解决阵面体法的单元形成问题。     

Heart rate variability: Linear and non-linear analysis of pre-awake period for normal and intrauterine growth restricted children at 10 year

Non-optimal fetal growth has been associated with an increased risk of coronary heart diseases in later life. Heart rate variability (HRV) is a non-invasive method reflecting autonomic cardiac function and decreased heart rate variability has been associated with arrhythmic complications in humans. This study compares the result of linear and non-linear HRV measures performed in long term (24 h) vs short term (15 min before awakening) interbeat interval time series data of normal and growth restricted children who were 9–10 years old. The aim of the study was to investigate which HRV measures obtained from short term recording reliably reflect information provided by long term recording. The comparability of HRV parameters derived from long term and short term recordings showed that low birth weight growth restricted children (IUGR) have low HRV. The findings indicated that low birth weight in growth retarded may be associated with negative cardiovascular outcome.     

Temperature-controlled microscopy for imaging living cells: apparatus, thermal analysis and temperature dependency of embryonic elongation in Caenorhabditis elegans

A new experimental apparatus for temperature-controlled microscopy has been developed for the study of the temperature dependency of developmental processes in the nematode Caenorhabditis elegans . However, the application of this apparatus is rather general and can be used for a wide range of temperatures between − 10 and 90 °C. The new apparatus is easy to use, inexpensive, simple to construct, and is designed for precise temperature control of oil-immersion microscopy using epifluorescence. Thermal analysis of the experimental apparatus shows the effects of each of its components, as well as the effects of uncertainty in temperature measurements. Finally, results of this study indicate that: (i) embryos incubated and imaged at temperatures of 8 °C and below do not elongate; (ii) the initial elongation rate is strongly temperature-dependent between 9 and 25 °C.     

Adsorbate deformation as a contrast mechanism in STM images of biopolymers in an aqueous environment: images of the unstained,hydrated DNA double helix

A stable residual aggregate remains on a submerged gold surface after electrophoretic deposition of DNA. We present scanning tunnelling microscope (STM) images of these aggregates which show many objects with the geometry of DNA, clearly displaying the 3·4 nm helix pitch. These images are quite distinctive, and cannot be generated when the deposition technique is used without DNA in the buffer solution. A characteristic of these images is that the tip is observed to dip down over the DNA molecule at the same time as the apparent barrier height drops by a factor of about four. The tip displacement is accounted for by a model in which contrast is dominated by local fluctuations in the deformability of the adsorbate layer, a quantity deduced from measurements of the apparent barrier heights in air, water, over small molecule adsorbates, and over DNA.     

Dynamics of subcellular organelles, growth hormone, Rab3B, SNAP-25, and syntaxin in rat pituitary cells caused by growth hormone releasing hormone and somatostatin

Rab3B is involved in the exocytosis of synaptic vesicles and secretory granules in the central nervous system and the anterior pituitary cells. The aim of this study was to elucidate both the role of rab3B in GH secretion and the mutual relationship of rab3B and SNARE proteins. Adult male rats were injected intravenously with 10 microg of growth hormone releasing hormone (GHRH) or 10 microg of somatostatin (SRIF). Untreated rats were used as controls, and their pituitary glands were sectioned for histochemical examination. Rab3B is localized on the limiting membrane of the secretory granules and the cytosol. Confocal laser scanning microscopic observation of immunohistochemical double staining of rab3B and GH revealed that immunoreactivity of rab3B increased in GHRH-treated rats and decreased in SRIF-treated rats. Confocal laser scanning microscopic observation of immunohistochemical double staining of SNAP-25, syntaxin, and rab3B revealed the co-localization of rab3B and these SNARE proteins in GHRH-treated rats, and their dissociation in SRIF-treated rats. These results suggest that rab3B plays a principal role in GH secretion in the anterior pituitary cells and that SNAP-25 and syntaxin act as co-workers with rab3B in the functional regulation of GH secretion.     

“Magic size” effect in the packing of n-alkanes on Au(1 1 1): evidence of lowered sliding force for molecules with specific length

Molecular structure of monolayers formed at the interface between Au(1 1 1) surfaces and solutions containing n-alkanes has been studied by in situ scanning tunneling microscopy at room temperature. Increasing the C_nH_2n+2 length from n=10 up to 50 with even n numbers alternates rectangular and tilted arrangement of alkanes within the self-organized layers. This alternation is related to the dramatically lowered sliding force for molecules with a length close to mT (m-integer), where T is the period of commensurability between the CH₂-CH₂-CH₂ period along alkyl chains and the interatomic distance along Au〈1 1 0〉 direction.     

Focal adhesion interactions with topographical structures: a novel method for immuno-SEM labelling of focal adhesions in S-phase cells

Current understanding of the mechanisms involved in osseointegration following implantation of a biomaterial has led to adhesion quantification being implemented as an assay of cytocompatibility. Such measurement can be hindered by intra-sample variation owing to morphological changes associated with the cell cycle. Here we report on a new scanning electron microscopical method for the simultaneous immunogold labelling of cellular focal adhesions and S-phase nuclei identified by BrdU incorporation. Prior to labelling, cellular membranes are removed by tritonization and antigens of non-interest blocked by serum incubation. Adhesion plaque–associated vinculin and S-phase nuclei were both separately labelled with a 1.4 nm gold colloid and visualized by subsequent colloid enhancement via silver deposition. This study is specifically concerned with the effects microgroove topographies have on adhesion formation in S-phase osteoblasts. By combining backscattered electron (BSE) imaging with secondary electron (SE) imaging it was possible to visualize S-phase nuclei and the immunogold-labelled adhesion sites in one energy 'plane' and the underlying nanotopography in another. Osteoblast adhesion to these nanotopographies was ascertained by quantification of adhesion complex formation.     

The switch mechanism of the cell death mode from apoptosis to necrosis in menadione-treated human osteosarcoma cell line 143B cells

Time-dependent changes in the cell death mode from apoptosis to necrosis were studied in cultured 143B cells treated with menadione, an anti-cancerous drug, excluding a possible involvement of "secondary necrosis." The population of apoptotic cells judged by FITC-Annexin V and propidium iodide (PI) double staining reached its maximum at 6 hours after 100 microM menadione treatment followed by an abrupt decrease thereafter, while that of necrotic cells continuously increased reaching 90% at 24 hours. Electron microscopically, cells attached to the culture dish at 6 hours after the treatment consisted of two different types of cells: cells with typical apoptotic features occupying the major population and those with condensed nuclei and swollen cytoplasm. Cells attached to the culture dish at 8 hours after the treatment consisted exclusively of those with condensed nuclei and swollen cytoplasm. Mitochondria in these cells showed various structural changes: those swollen to various degrees with deposition of flocculent densities, or those with highly condensed matrix. Distinct decreases both in intracellular levels of ATP and caspase-3-like activities and remarkable elevations of intracellular levels of superoxide, which were partly suppressed by NAD(P)H oxidase inhibitors, occurred at 6 hours after the treatment. These results may suggest that distinct increases of the intracellular level of superoxide derived from plasma membrane NAD(P)H oxidase besides that from mitochondria have triggered the transition of cell death mode from apoptosis to necrosis. Transition of highly condensed mitochondria to extremely swollen ones may reflect necrotic processes in menadione-treated cells. The present study strongly suggests that time-dependent study is essential using the electron microscopic technique to analyze detailed processes in the changes of the cell death mode.