Morphology, mechanical properties and viability of encapsulated cells

The morphological and mechanical properties of encapsulated yeast cells (Saccharomyces cerevisiae) have been investigated by atomic force microscopy (AFM). Single living cells have been coated through the alternate deposition of oppositely charged polyelectrolyte (PE) layers. The properties of cells coated by different numbers of PE layers and from PE solutions of different ionic strength have been investigated. AFM imaging indicates an increase in PE coating stability when decreasing the solution ionic strength. The Young's moduli of the different examined systems have been evaluated through a quantitative analysis of force-distance curves by using the Hertz-Sneddon model. The analysis indicates an increase in hybrid system stiffness when lowering the ionic strength of the PE solution. An evaluation of the viability of encapsulated cells was obtained by confocal laser scanning microscopy (CLSM) measurements. CLSM analysis indicates that cells preserve their subcellular structure and duplication capability after encapsulation. By coupling AFM and CLSM data, a correlation between local stiffness and duplication rate was obtained.     

Saccharomyces cerevisiae samples stained with FUN-1 dye can be stored at −20°C for later observation

FUN‐1, a fluorescent vital dye, has been observed to form cylindrical intravacuolar structures within the vacuoles of metabolically active yeast cells. FUN‐1 staining, which begins as a diffuse pool of fluorescent cytoplasmic stain, uses an unknown endogenous biochemical processing mechanism to compact and form orange‐red cylindrical intravacuolar structures within the cell vacuole. In the clinical setting, FUN‐1 is primarily used for identification of fungal infection. FUN‐1 is utilized in the laboratory to distinguish between metabolically active and dead fungal cells. Although this stain is useful for distinguishing between live and dead fungal dead cells, few studies have utilized this chemical. This lack of use in the scientific community may be due to the requirement that cells are visualized directly after staining. Thus, it would be of interest to be able to stain cells and store them for later use. Our lab examined the longevity of cylindrical intravacuolar structures in two strains of Saccharomyces cerevisiae stained with FUN‐1 and stored at −20°C. We found that cylindrical intravacuolar structures could be reliably observed and imaged utilizing differential interference contrast microscopy and fluorescence microscopy for 21 days. We also observed that cells stained with FUN‐1 would resume propagation on yeast extract, peptone, dextrose (YPD) plates after being frozen at −20°C for 21 days. These modifications to the published procedure for FUN‐1 dye staining should allow for a more prevalent and less time sensitive use of this important biological tool.     

Morphological and functional characterization of circulating hemocytes using microscopy techniques

In the present study, Microscopy studies were performed to characterize the blood cells of the mangrove crab Episesarma tetragonum. Three types of hemocytes were observed: granulocytes, semi‐granulocytes, and hyalinocytes or agranulocytes. Hyalinocytes have a distinguished nucleus surrounded by the cytoplasm, and a peculiar cell type was present throughout the cytosol, lysosomes with hemocyte types (granules) stained red (pink). Giemsa staining was used to differentiate between the large and small hemocytes. Ehrlich's staining was used to differentiate granule‐containing cells in acidophils (55%), basophils (44%), and neutrophils (<1%). Periodic acid–Schiff staining was used to identify the sugar molecules in the cytoplasm. Cell‐mediated immune reactions including phagocytosis, encapsulation, agglutination, and peroxidase‐mediated cell adhesion are the functions of hemocytes. Agglutination reaction involves both kind of cells involved in yeast and heme‐agglutination responses in invertebrates. The beta glucan outer layer of yeast cells was recognized by hemocyte receptors. Human RBC cells were agglutinated via granulocytes. E. tetragonum hemocytes are an important animal model for studying both ultrastructural and functional activity of circulating cells. In addition, E. tetragonum hemocytes exhibited excellent antibacterial and antibiofilm activities were studied through plating and microplate assays. Biofilm inhibition was also visualized through changes in biochemical assays and morphological variations were visualized through levels in in situ microscopy analysis.     

A polyaniline based intrinsically conducting coating for corrosion protection of structural steels

Among the various corrosion protection strategies for structural steels, coating techniques provide the most cost‐effective protection and have been used as the primary mode of corrosion protection. Existing coating techniques however have been used mainly for their barrier capability and therefore all have a limited service life due to oxidation aging, electrolytic degradation, or various inadvertent defects and flaws occurred in and after coating applications. This work investigated the anti‐corrosion potential of a π‐conjugated polymer—polyaniline (PANi), which was doped into an intrinsically conducting polymer and then included in a two‐layer coating system as a primer layer. To achieve a long service life, the primer layer was made by mixing the conductive PANi in a waterborne poly‐vinyl butyral solution to provide strong adhesion to steel surface, and then topcoated with a layer of elastomer‐modified polyethylene to obtain extra mechanical and barrier protections. Two ASTM standard tests were conducted to evaluate the corrosion durability and tensile adhesion of the two‐layer system, in which the system demonstrated superior performance. The Scanning Kelvin Probe Force Microscopy (SKPFM) was used to provide the microscopic evidences for the outstanding performance. Microsc. Res. Tech. 76:1186–1195, 2013. © 2013 Wiley Periodicals, Inc.     

Correlative scanning electron and confocal microscopy imaging of labeled cells coated by indium‐tin‐oxide

Confocal microscopy imaging of cells allows to visualize the presence of specific antigens by using fluorescent tags or fluorescent proteins, with resolution of few hundreds of nanometers, providing their localization in a large field‐of‐view and the understanding of their cellular function. Conversely, in scanning electron microscopy (SEM), the surface morphology of cells is imaged down to nanometer scale using secondary electrons. Combining both imaging techniques have brought to the correlative light and electron microscopy, contributing to investigate the existing relationships between biological surface structures and functions. Furthermore, in SEM, backscattered electrons (BSE) can image local compositional differences, like those due to nanosized gold particles labeling cellular surface antigens. To perform SEM imaging of cells, they could be grown on conducting substrates, but obtaining images of limited quality. Alternatively, they could be rendered electrically conductive, coating them with a thin metal layer. However, when BSE are collected to detect gold‐labeled surface antigens, heavy metals cannot be used as coating material, as they would mask the BSE signal produced by the markers. Cell surface could be then coated with a thin layer of chromium, but this results in a loss of conductivity due to the fast chromium oxidation, if the samples come in contact with air. In order to overcome these major limitations, a thin layer of indium‐tin‐oxide was deposited by ion‐sputtering on gold‐decorated HeLa cells and neurons. Indium‐tin‐oxide was able to provide stable electrical conductivity and preservation of the BSE signal coming from the gold‐conjugated markers. Microsc. Res. Tech. 78:433–443, 2015. © 2015 Wiley Periodicals, Inc.     

Magnetosomal matrix: ultrafine structure may template biomineralization of magnetosomes

The organic matrix surrounding bullet‐shaped, cubo‐octahedral, D‐shaped, irregular arrowhead‐shaped, and truncated hexa‐octahedral magnetosomes was analysed in a variety of uncultured magnetotactic bacteria. The matrix was examined using low‐ (80 kV) and intermediate‐ (400 kV) voltage TEM. It encapsulated magnetosomes in dehydrated cells, ultraviolet‐B‐irradiated dehydrated cells and stained resin‐embedded fixed cells, so the apparent structure of the matrix does not appear to be an artefact of specimen preparation. High‐resolution images revealed lattice fringes in the matrix surrounding magnetite and greigite magnetosomes that were aligned with lattice fringes in the encapsulated magnetosomes. In all except one case, the lattice fringes had widths equal to or twice the width of the corresponding lattice fringes in the magnetosomes. The lattice fringes in the matrix were aligned with the {311}, {220}, {331}, {111} and {391} related lattice planes of magnetite and the {222} lattice plane of greigite. An unidentified material, possibly an iron hydroxide, was detected in two immature magnetosomes containing magnetite. The unidentified phase had a structure similar to that of the matrix as it contained {311}, {220} and {111} lattice fringes, which indicates that the matrix acts as a template for the spatially controlled biomineralization of the unidentified phase, which itself transforms into magnetite. The unidentified phase was thus called pre‐magnetite. The presence of the magnetosomal matrix explains all of the five properties of the biosignature of the magnetosomal chain proposed previously by Friedmann et al. and supports their claim that some of the magnetite particles in the carbonate globules in the Martian meteorite ALH84001 are biogenic. Two new morphologies of magnetite magnetosomes are also reported here (i.e. tooth‐shaped and hexa‐octahedral magnetosomes). Tooth‐shaped magnetite magnetosomes elongated in the [110] direction are reported, and are distinct from arrowhead‐shaped and bullet‐shaped magnetosomes. Elongation of magnetite magnetosomes in the [110] direction has not been reported previously. A Martian hexa‐octahedral magnetite particle was previously characterized by Thomas‐Keptra et al. and compared with truncated hexa‐octahedral magnetite magnetosomes. Hexa‐octahedral magnetite magnetosomes with the same morphology and similar sizes and axial ratios as those reported by Thomas‐Keptra et al. are characterized here. These observations support their claim that ALH84001 contains evidence for a past Martian biota.     

Quantitative FRET measurement using emission‐spectral unmixing with independent excitation crosstalk correction

Quantification of fluorescence resonance energy transfer (FRET) needs at least two external samples, an acceptor‐only reference and a linked FRET reference, to calibrate fluorescence signal. Furthermore, all measurements for references and FRET samples must be performed under the same instrumental conditions. Based on a novel notion to predetermine the molar extinction coefficient ratio (R_C) of acceptor‐to‐donor for the correction of acceptor excitation crosstalk, we present here a robust and independent emission‐spectral unmixing FRET methodology, Iem‐spFRET, which can simultaneously measure the E and R_C of FRET sample without any external references, such that Iem‐spFRET circumvents the rigorous restriction of keeping the same imaging conditions for all FRET experiments and thus can be used for the direct measurement of FRET sample. We validate Iem‐spFRET by measuring the absolute E and R_C values of standard constructs with different acceptor‐to‐donor stoichiometry expressed in living cells. Our results demonstrate that Iem‐spFRET is a simple and powerful tool for real‐time monitoring the dynamic intermolecular interaction within single living cells.     

High-resolution scanning electron microscopy of immunogold-labelled cells by the use of thin plasma coating of osmium

The feasibility of plasma coating of a thin osmium layer for high‐resolution immuno‐scanning electron microscopy of cell surfaces was tested, using Drosophila embryonic motor neurones as a model system. The neuro‐muscular preparations were fixed with formaldehyde and labelled with a neurone‐specific antibody and 10 or 5 nm colloidal gold‐conjugated secondary antibodies. The specimens were post‐fixed with osmium tetroxide and freeze‐dried. Then they were coated with a 1–2 nm thick layer of osmium using a hollow cathode plasma coater. The thin and continuous coating of amorphous osmium gave good signals of gold particles and fine surface structures of neurites in backscattered electron images simultaneously. This method makes it possible to visualize the antigen distribution and the three‐dimensionally complex surface structures of cellular processes with a resolution of several nanometres.     

The beauty of the yeast: live cell microscopy at the limits of optical resolution

The yeast Saccharomyces cerevisiae is a very powerful system for cell biological research. Recent advances in electronic light microscopy together with the application of green fluorescent protein and other in vivo staining techniques have allowed novel and exciting insights into structural organization and dynamics of cells as small as yeast. Methods for staining yeast for microscopic inspection and for introducing tags for localization studies of proteins in living or fixed cells are summarized. Electronic light microscopy, video/deconvolution methods, and confocal laser scanning microscopy as novel tools for structural analyses, and their practical applications in yeast, are discussed.     

Shear bond strength and ultrastructural interface analysis of different adhesive systems to bleached dentin

Background: It remains unclear as to whether or not dental bleaching affects the bond strength of dentin/resin restoration. Purpose: To evaluated the bond strength of adhesive systems to dentin submitted to bleaching with 38% hydrogen peroxide (HP) activated by LED‐laser and to assess the adhesive/dentin interfaces by means of SEM. Study design: Sixty fragments of dentin (25 mm²) were included and divided into two groups: bleached and unbleached. HP was applied for 20 s and photoactivated for 45 s. Groups were subdivided according to the adhesive systems (n = 10): (1) two‐steps conventional system (Adper Single Bond), (2) two‐steps self‐etching system (Clearfil standard error (SE) Bond), and (3) one‐step self‐etching system (Prompt L‐Pop). The specimens received the Z250 resin and, after 24 h, were submitted to the bond strength test. Additional 30 dentin fragments (n = 5) received the same surface treatments and were prepared for SEM. Data were analyzed by ANOVA and Tukey's test (α = 0.05). Results: There was significant strength reduction in bleached group when compared to unbleached group (P < 0.05). Higher bond strength was observed for Prompt. Single Bond and Clearfil presented the smallest values when used in bleached dentin. SEM analysis of the unbleached specimens revealed long tags and uniform hybrid layer for all adhesives. In bleached dentin, Single Bond provided open tubules and with few tags, Clearfil determined the absence of tags and hybrid layer, and Prompt promoted a regular hybrid layer with some tags. Conclusions: Prompt promoted higher shear bond strength, regardless of the bleaching treatment and allowed the formation of a regular and fine hybrid layer with less deep tags, when compared to Single Bond and Clearfil. Microsc. Res. Tech., 2010. © 2010 Wiley‐Liss, Inc.     

General morphology and ultrastructure of the female reproductive apparatus of Trichomalopsis shirakii crawford (Hymenoptera,Pteromalidae)

The morphology and ultrastructure of the female reproductive system were examined for a larval–pupal parasitoid Trichomalopsis shirakii Crawford of Oulema oryzae Kuwayama using light and electron microscopes. The reproductive system includes two ovaries, two pairs of accessory glands, an unbranched venom gland, a large venom reservoir and a Dufour gland. Each ovariole contains follicles and oocytes at different stages of maturation. A fibrous layer covers the surface of mature egg. The accessory glands are made up of a layer of secretory cells surrounded by muscle fibers. In these secretory cells, numerous mitochondria, electron‐dense secretory granules and vesicles filled with dense granular particles are present. These granular particles appear as virus‐like particles (VLPs). The venom gland consists of a single layer of secretory cells which are organelle rich with abundant rough endoplasmic reticulum, mitochondria and vesicular organelles, a layer of duct cells and an inner intima. The reservoir consists of a muscular sheath, epidermal cells with few organelles and an intima layer. The Dufour gland has a relatively large lumen surrounded by a single layer of columnar epithelial cells which are characterized by clusters of smooth endoplasmic reticulum and lipid droplets. Aside from the venom, the fibrous layer coating the egg and the granular particles which may be VLPs have been discovered in our study. They may serve as one of the parasitoid‐associated factors in their host–parasitoid relationship and play a role in host immune suppression. Microsc. Res. Tech. 79:625–636, 2016. © 2016 Wiley Periodicals, Inc.     

A novel whole tooth-in-jaw-bone culture of rat molars: Morphological, immunohistochemical, and laser capture microdissection analysis

In conventional whole‐tooth culture systems, limitation exists regarding maintenance of the vitality of the dental pulp, because this tissue is encased in rigid dentin walls that hinder nutrition supply. We here report a whole tooth‐in‐jaw‐bone culture system of rat mandibular first molars, where transcardiac perfusion with culture medium was carried out before placement of the jaw bone into culture medium, aiming to facilitate longer time preservation of the dental pulp tissue. Following 7 days of culture, the pulp tissues were analyzed by histology and immunohistochemistry to ED2 (antiresident macrophage). ED2‐positive macrophages were also analyzed for their Class II MHC, interleukin‐6 (IL‐6), and p53 mRNA expression levels by means of immune‐laser capture microdissection (immune‐LCM). Dentin sialophosphoprotein (DSPP) mRNA expression in odontobalstic layer was also examined by LCM. Teeth cultured following saline‐perfusion and nonperfusion served as cultured controls. Normal teeth also served as noncultured controls. Histological examination demonstrated that the structure of the pulp tissue was well preserved in the medium‐perfused explants in contrast to the cultured control groups. The Class II MHC, IL‐6, and p53 mRNA expression levels of ED2‐positive cells and DSPP expression levels of odontoblastic layer tissues in the pulp of medium‐perfused explants were not significantly different from those in the noncultured normal teeth. In conclusion, the structural integrity and mRNA expression in the pulp were maintained at the in vivo level in the ex vivo whole tooth‐in‐jaw‐bone culture system. The system may lay the foundation for studies aiming at defining further histological and molecular mechanism of the pulp. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.     

Real‐time histological imaging of kidneys stained with food dyes using multiphoton microscopy

We have developed a real‐time imaging technique for diagnosis of kidney diseases which is composed of two steps, staining renal cells safely with food dyes and optical sectioning of living renal tissue to obtain histological images by multiphoton microscopy (MPM). Here, we demonstrated that the MPM imaging with food dyes, including erythrosine and indigo carmine, could be used as fluorescent agents to visualize renal functions and structures such as glomerular bloodstreams, glomerular filtration, and morphology of glomeruli and renal tubules. We also showed that the kidneys of IgA nephropathy model‐mice stained with the food dyes presented histopathological characteristics different from those observed in normal kidneys. The use of the food dyes enhances the quality of tissue images obtained by MPM and offers the potential to contribute to a clinical real‐time diagnosis of kidney diseases. Microsc. Res. Tech. 78:847–858, 2015. © 2015 Wiley Periodicals, Inc.     

Behavior of Coated Carbide Tools in High Speed Machining of a Nickel Base Alloy

Multilayer TiN/TiCN/TiN and single-layer TiAIN PVD coated carbide tools were used to machine a nickel base, C-263, alloy at high-speed conditions in order to investigate their performance in terms of tool life, surface finish and component forces generated during machining. The test results show that the triple layer, TiN/TiCN/TiN, coated inserts gave longer tool life when machining at higher speed and depth of cut conditions while the single layer, TiA/N, coated inserts produced better surface finish. The feed forces recorded were generally higher than the cutting forces. This could perhaps be attributed to the adverse effect of burr formation and work hardening of the workpiece associated with prolonged machining. Analysis of the test results indicate that the difference in thermal properties and tribo-chemical behaviour of both the coating and substrate materials are the major factors influencing the tribo-contact at the tool-chip interface during machining. Wear mechanisms of the coating materials can also affect tool performance in terms of tool life, surface finish and component forces.     

A new rapid modification for membrane fluorescence tests based on the Microtiter system.

This paper describes the micro-membrane-fluorescence test (MMF), which is a new semiautomated technique employing units of the Microtiter system. This method allowing the performance of several hundred samples within a few hours may contribute to further standardization of immunofluorescence work on the cellular level. Furthermore, it can also be applied in immunofluorescence tests were antigen coated particles (beads) are used as antigenic substrate, instead of living cells.     

Multichannel wide‐field microscopic FRET imaging based on simultaneous spectral unmixing of excitation and emission spectra

Simultaneous spectral unmixing of excitation and emission spectra (ExEm unmixing) has inherent ability resolving spectral crosstalks, two key issues of quantitative fluorescence resonance energy transfer (FRET) measurement, of both the excitation and emission spectra between donor and acceptor without additional corrections. We here set up a filter‐based multichannel wide‐field microscope for ExEm unmixing‐based FRET imaging (m‐ExEm‐spFRET) containing a constant system correction factor (f_sc) for a stable system. We performed m‐ExEm‐spFRET with four‐ and two‐wavelength excitation respectively on our system to quantitatively image single living cells expressing FRET tandem constructs, and obtained accurate FRET efficiency (E) and concentration ratio of acceptor to donor (R_C). We also performed m‐ExEm‐spFRET imaging for single living cells coexpressing CFP‐Bax and YFP‐Bax, and found that the E values were about 0 for control cells and about 28% for staurosporin‐treated cells when R_C were larger than 1, indicating that staurosporin induced significant oligomerisation.     

Friction properties of perfluorinated polyethers for hot‐dipped tin low‐level separable electrical contacts

Tin coatings are used as a final protection for cuprous substrates involved in low‐level electrical contacts. High friction forces and fretting phenomena remain a cause of electrical failure of connector terminals, and so should be minimised. A lubricant layer can improve the life and reliability of connector terminals but such a layer must ensure low and stable resistance values even under severe temperature conditions. Work has been undertaken to investigate the lubricant layer most suitable for the protection of tinned electrical contacts. Perfluorinated polyethers (PFPEs) were chosen for their inertness. The aim was to correlate various physico‐chemical properties of branched or linear PFPEs, with their frictional and electrical properties. The long‐term electrical properties of a contact are shown to be linked to the initial wear mode in the contact.     

Light sheet based imaging flow cytometry on a microfluidic platform

We propose a light sheet based imaging flow cytometry technique for simultaneous counting and imaging of cells on a microfluidic platform. Light sheet covers the entire microfluidic channel and thus omits the necessity of flow focusing and point scanning based technology. Another advantage lies in the orthogonal detection geometry that totally cuts‐off the incident light, thereby substantially reducing the background in the detection. Compared to the existing state‐of‐art techniques the proposed technique shows marked improvement. Using fluorescently‐coated Saccharomyces cerevisiae cells we have recorded cell counting with throughput as high as 2,090 cells/min in the low flow rate regime and were able to image the individual cells on‐the‐go. Overall, the proposed system is cost‐effective and simple in channel geometry with the advantage of efficient counting in operational regime of low laminar flow. This technique may advance the emerging field of microfluidic based cytometry for applications in nanomedicine and point of care diagnostics. Microsc. Res. Tech. 76:1101–1107, 2013. © 2013 Wiley Periodicals, Inc.     

Direct visualization of receptor arrays in frozen-hydrated sections and plunge-frozen specimens of E. coli engineered to overproduce the chemotaxis receptor Tsr

We have recently reported electron tomographic studies of sections obtained from chemically fixed E. coli cells overproducing the 60‐kDa chemotaxis receptor Tsr. Membrane extracts from these cells prepared in the presence of Tween‐80 display hexagonally close‐packed microcrystalline assemblies of Tsr, with a repeating unit large enough to accommodate six Tsr molecules arranged as trimers of receptor dimers. Here, we report the direct visualization of the Tsr receptor clusters in (i) vitrified cell suspensions of cells overproducing Tsr, prepared by rapid plunge‐freezing, and (ii) frozen‐hydrated sections obtained from cells frozen under high pressure. The frozen‐hydrated sections were generated by sectioning at ?150 °C using a diamond knife with a 25° knife angle, with nominal thicknesses ranging from 20 to 60 nm. There is excellent correspondence between the spatial arrangement of receptors in thin frozen‐hydrated sections and the arrangements found in negatively stained membrane extracts and plunge‐frozen cells, highlighting the potential of using frozen‐hydrated sections for the study of macromolecular assemblies within cells under near‐native conditions.