Tau binds ATP and induces its aggregation

Tau is a microtubule‐associated protein mainly found in neurons. The protein is associated with process of microtubule assembly, which plays an important role in intracellular transport and cell structure of the neuron. Tauopathies are a group of neurodegenerative diseases specifically associated with tau abnormalities. While a well‐defined mechanism remains unknown, most facts point to tau as a prominent culprit in neurodegeneration. In most cases of Tauopathies, aggregates of hyperphosphorylated tau have been found. Two proposals are present when discussing tau toxicity, one being the aggregation of tau proteins and the other points toward a conformational change within the protein. Previous work we carried out showed tau hyperphosphorylation promotes tau to behave abnormally resulting in microtubule assembly disruption as well as a breakdown in tau self‐assembly. We found that tau's N‐terminal region has a putative site for ATP/GTP binding. In this paper we demonstrate that tau is able to bind ATP and not GTP, that this binding induces tau self‐assembly into filaments. At 1 mM ATP the filaments are 4–7 nm in width, whereas at 10 mM ATP the filaments appeared to establish lateral interaction, bundling and twisting, forming filaments that resembled the Paired Helical Filaments (PHF) isolated from Alzheimer disease brain. ATP‐induced self‐assembly is not energy dependent because the nonhydrolysable analogue of the ATP induces the same assembly. Microsc. Res. Tech. 77:133–137, 2014. © 2013 Wiley Periodicals, Inc.     

Influence of double application technique on the bonding effectiveness of self‐etch adhesive systems

Aim: To evaluate and compare the effect of double‐application of single‐step self‐etch adhesives using microleakage study and to analyze the dentin–adhesive interfacial micromorphology. Methods: In total, 72 extracted human premolars were divided into three groups for different self‐etch adhesives (G Bond, GC [GB], Optibond, Kerr [OB], and Xeno V Plus, Dentsply [XV]). Class V cavities were prepared. Each group was further divided into two subgroups (n = 10) according to the placement technique of the adhesive, using the single‐application [subgroup (a)] or double‐application method [subgroup (b)]. Resin composite (Z 250, 3M ESPE, St. Paul, MN) was used to restore the cavities and light cured for 40 s. Twenty samples from each group were subjected to microleakage study. Two samples from both the subgroups of the three adhesives were used for scanning electron microscopic examination of the resin–dentin interfacial ultrastructure. Dye leakage scores were subjected to statistical analysis using Kruskal–Wallis and Mann–Whitney U‐tests at significance level of P < 0.05. Results: GB depicted significantly more microleakage which was significantly greater than OB and XV. The double application led to significant decrease in microleakage of GB with no significant effect on the microleakage scores of other two all‐in‐one adhesives, that is OB and XV. Conclusion: Double application of all‐in‐one self‐etch adhesives improves the marginal sealing ability in dentin although it appears to be product dependent. Microsc. Res. Tech. 78:489–494, 2015. © 2015 Wiley Periodicals, Inc.     

MRT Letter: Two‐photon excitation‐based 2pi Light‐sheet system for nano‐lithography

We propose two‐photon excitation‐based light‐sheet technique for nano‐lithography. The system consists of 2 ‐configured cylindrical lens system with a common geometrical focus. Upon superposition, the phase‐matched counter‐propagating light‐sheets result in the generation of identical and equi spaced nano‐bump pattern. Study shows a feature size of as small as few tens of nanometers with a inter‐bump distance of few hundred nanometers. This technique overcomes some of the limitations of existing nano‐lithography techniques, thereby, may pave the way for mass‐production of nano‐structures. Potential applications can also be found in optical microscopy, plasmonics, and nano‐electronics. Microsc. Res. Tech. 78:1–7, 2015. © 2014 Wiley Periodicals, Inc.     

Microstructural analysis of iron aluminide formed by self-propagating high-temperature synthesis mechanism in aluminium matrix composite

An aluminium matrix composite with iron aluminide formed in situ as a result of self‐propagated high‐temperature synthesis was examined. The structural characteristics of the reinforcement investigated by scanning electron microscopy and transmission electron microscopy methods are presented. Iron aluminide particles with a very fine grain size and of two shapes, cubic and needle‐like, were observed. No differences in their phase composition were found by the selective electron diffraction pattern method. The composite reinforcement formed in the early stage of self‐propagating high‐temperature synthesis consisted only of the Al₃Fe phase.     

Cryogenic‐temperature electron microscopy direct imaging of carbon nanotubes and graphene solutions in superacids

Cryogenic electron microscopy (cryo‐EM) is a powerful tool for imaging liquid and semiliquid systems. While cryogenic transmission electron microscopy (cryo‐TEM) is a standard technique in many fields, cryogenic scanning electron microscopy (cryo‐SEM) is still not that widely used and is far less developed. The vast majority of systems under investigation by cryo‐EM involve either water or organic components. In this paper, we introduce the use of novel cryo‐TEM and cryo‐SEM specimen preparation and imaging methodologies, suitable for highly acidic and very reactive systems. Both preserve the native nanostructure in the system, while not harming the expensive equipment or the user. We present examples of direct imaging of single‐walled, multiwalled carbon nanotubes and graphene, dissolved in chlorosulfonic acid and oleum. Moreover, we demonstrate the ability of these new cryo‐TEM and cryo‐SEM methodologies to follow phase transitions in carbon nanotube (CNT)/superacid systems, starting from dilute solutions up to the concentrated nematic liquid‐crystalline CNT phases, used as the ‘dope’ for all‐carbon‐fibre spinning. Originally developed for direct imaging of CNTs and graphene dissolution and self‐assembly in superacids, these methodologies can be implemented for a variety of highly acidic systems, paving a way for a new field of nonaqueous cryogenic electron microscopy.     

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.     

Super‐resolution optical microscopy based on scannable cantilever‐combined microsphere

We report an ingenious method of super‐resolution optical microscopy utilizing scannable cantilever‐combined microsphere. By scanning the microsphere over the sample surface in a cantilever‐combined microsphere‐sample contact state, super‐resolution images can be acquired at arbitrary sample regions through near‐field information collection by the microsphere. In addition, such a state can effectively reduce the possibility of breaking the cantilever and damaging the microsphere or sample surface. This work has developed a new method and technique of sub‐diffraction‐limit optical microscopy, and can be practically applied in various fields of micro/nanoscopy. Microsc. Res. Tech. 78:1128–1132, 2015. © 2015 Wiley Periodicals, Inc.     

Phasor‐FLIM analysis of Thioflavin T self‐quenching in Concanavalin amyloid fibrils

The formation of amyloid structures has traditionally been related to human neurodegenerative pathologies and, in recent years, the interest in these highly stable nanostructures was extended to biomaterial sciences. A common method to monitor amyloid growth is the analysis of Thioflavin T fluorescence. The use of this highly selective dye, diffused worldwide, allows mechanistic studies of supramolecular assemblies also giving back important insight on the structure of these aggregates. Here we present experimental evidence of self‐quenching effect of Thioflavin T in presence of amyloid fibrils. A significant reduction of fluorescence lifetime of this dye which is not related to the properties of analyzed amyloid structures is found. This result is achieved by coupling Fluorescence Lifetime Imaging Microscopy with phasor approach as suitable model‐free methods and constitute a serious warning that have to be taken in account if is dye is used for quantitative studies.     

Three‐dimensional Co‐culture of hepatic progenitor cells and mesenchymal stem cells in vitro and in vivo

Introduction: Here we co‐cultured hepatic progenitor cells (HPCs) and mesenchymal stem cells (MSCs) to investigate whether the co‐culture environments could increase hepatocytes form. Methods: Three‐dimensional (3D) co‐culture model of HPCs and MSCs was developed and morphological features of cells were continuously observed. Hepatocyte specific markers Pou5f1/Oct4, AFP, CK‐18 and Alb were analyzed to confirm the differentiation of HPCs. The mRNA expression of CK‐18 and Alb was analyzed by RT‐PCR to investigate the influence of co‐culture model to the terminal differentiation process of mature hepatocytes. The functional properties of hepatocyte‐like cells were detected by continuously monitoring the albumin secretion using Gaussia luciferase assays. Scaffolds with HPCs and MSCs were implanted into nude mouse subcutaneously to set up the in vivo co‐culture model. Results: Although two groups formed smooth spheroids and high expressed of CK‐18 and Alb, hybrid spheroids had more regular structures and higher cell density. CK‐18 and Alb mRNA were at a relatively higher expression level in co‐culture system during the whole cultivation time (P < 0.05). Albumin secretion rates in the hybrid spheroids had been consistently higher than that in the mono‐culture spheroids (P < 0.05). In vivo, the hepatocyte‐like cells were consistent with the morphological features of mature hepatocytes and more well‐differentiated hepatocyte‐like cells were observed in the co‐culture group. Conclusions: HPCs and MSCs co‐culture system is an efficient way to form well‐differentiated hepatocyte‐like cells, hence, may be helpful to the cell therapy of hepatic tissues and alleviate the problem of hepatocytes shortage. Microsc. Res. Tech. 78:688–696, 2015. © 2015 Wiley Periodicals, Inc.     

Analysis of efficacy of the self‐assembling peptide‐based remineralization agent on artificial enamel lesions

The objective of this in vitro study was to analyze and compare the biomimetic remineralizing efficacy of the self‐assembling peptide (P11‐4) with agents containing casein phoshopeptide‐amorhous calcium phosphate fluoride (CPP‐ACFP) and sodium fluoride (NaF) on artificial caries lesions using DIAGNOdent and micro‐computed tomography (μCT). Artificial enamel lesions were prepared on extracted impacted sound mandibular third molars. The samples were randomly allocated to four groups (n = 8): Group 1, P11‐4 (Curodont Repair, Credentis AG, Switzerland); Group 2, CPP‐ACFP (MI Varnish, GCCo., Japan); Group3, NaF (Duraphat Varnish, Colgate, Colgate‐Palmolive, NY, USA); Group 4, artificial saliva (control). The agents were applied to demineralized surfaces according to manufacturers' instructions; all specimens were stored in artificial saliva for 1 month. Demineralization and remineralization on enamel surfaces were analyzed and quantified by DIAGNOdent (KaVo, Germany) and μCT (SkyScan1174, Belgium) for lesion depth/area/volume/mineral density (MD). The remineralization efficacy of the agents was evaluated by DIAGNOdent on 1st, 7th, 30th days and by μCT on 30th day. Data were statistically analyzed by ANOVA, Kruskal–Wallis, T test, and Wilxocon tests. The highest remineralization efficacy findings in all periods were determined in Group 1, followed by Groups 2, 3, and 4. The remineralization findings for fluorescence, MD, lesion depth in Group 1 were found significantly higher (p < 0.01) than Group 3; and no significant differences (p > 0.05) were found between Groups 1–2 and Groups 2–3. The area and volume change values in Groups 1, 2, and 3 have shown no significancy (p > 0.05). A significant correlation (p < 0.01) was found between μCT and DIAGNOdent methods. The data of this study have demonstrated that P11‐4 has showed the best remineralization efficacy, followed by CPP‐ACFP and NaF. It is concluded that self‐assembling peptide‐based remineralization agent can be used successfully for biomimetic remineralization of enamel subsurface lesions.     

Internal architecture of coffin‐shaped ZSM‐5 zeolite crystals with hourglass contrast unravelled by focused ion beam‐assisted transmission electron microscopy

Optical microscopy, focused ion beam and transmission electron microscopy are combined to study the internal architecture in a coffin‐shaped ZSM‐5 crystal showing an hourglass contrast in optical microscopy. Based on parallel lamellas from different positions in the crystal, the orientation relationships between the intergrowth components of the crystal are studied and the internal architecture and growth mechanism are illustrated. The crystal is found to contain two pyramid‐like components aside from a central component. Both pyramid‐like components are rotated by 90° along the common c‐axis and with respect to the central component while the interfaces between the components show local zig‐zag feature, the latter indicating variations in relative growth velocity of the two components. The pyramid‐like intergrowth components are larger and come closer to one another in the middle of the crystal than at the edges, but they do not connect. A model of multisite nucleation and growth of 90° intergrowth components is proposed.     

Different bacterial models for in vitro induction of non‐cavitated enamel caries‐like lesions: Microhardness and polarized light miscroscopy analyses

The aim of this study was to compare different bacterial models for in vitro induction of non‐cavitated enamel caries‐like lesions by microhardness and polarized light microscopy analyses. One hundred blocks of bovine enamel were randomly divided into four groups (n = 25) according to the bacterial model for caries induction: (A) Streptococcus mutans, (B) S. mutans and Lactobacillus acidophilus, (C) S. mutans and L. casei, and (D) S. mutans, L. acidophilus, and L. casei. Within each group, the blocks were randomly divided into five subgroups according to the duration of the period of caries induction (4–20 days). The enamel blocks were immersed in cariogenic solution containing the microorganisms, which was changed every 48 h. Groups C and D presented lower surface hardness values (SMH) and higher area of hardness loss (ΔS) after the cariogenic challenge than groups A and B (P < 0.05). As regards lesion depth, under polarized light microscopy, group A presented significantly lower values, and groups C and D the highest values. Group B showed a higher value than group A (P < 0.05). Groups A and B exhibited subsurface caries lesions after all treatment durations, while groups C and D presented erosion‐type lesions with surface softening. The model using S. mutans, whether or not it was associated with L. acidophilus, was less aggressive and may be used for the induction of non‐cavitated enamel caries‐like lesions. The optimal period for inducing caries‐like lesions was 8 days. Microsc. Res. Tech. 78:444–451, 2015. © 2015 Wiley Periodicals, Inc.     

Assembly of Single-Stranded DNA Onto HOPG Surface at Different Temperature: Atomic Force Microscopy Study

Assembly of long single‐stranded DNA (ssDNA) and short oligodeoxynucleotides onto bare highly oriented pyrolytic graphite (HOPG) at different temperature has been studied. It was indicated that both long ssDNA and oligodeoxynucleotides can sequentially form network, straight chains, and layer structures when the adsorption temperature was changed from room temperature, 37–55°C. High‐resolution atomic force microscopy (AFM) imaging of the layer structures revealed that they are composed of parallel ssDNA chains with relatively higher height and tend to form patterns with three‐fold symmetry. These new findings are significantly important for understanding assembly characterization of ssDNA. In addition, this assembly method for ssDNA is expected to be used for preparation of DNA structures in biosensing and DNA‐based nanodevices. SCANNING 34: 302–308, 2012. © 2012 Wiley Periodicals, Inc.     

Direct atomic force microscopy observations of monovalent ion induced binding of DNA to mica

Multivalent ions in solution are known to mediate attraction between two like‐charged molecules. Such attraction has proved useful in atomic force microscopy (AFM) where DNA may be immobilized to a mica surface facilitating direct imaging in liquid. Theories of DNA immobilization suggest that either ‘salt bridging’ or fluctuation in the positions of counter ions about both the mica surface and DNA backbone secure DNA to the mica substrate. Whilst both theoretical and experimental evidence suggest that immobilization is possible in the presence of divalent ions, very few studies identify that such immobilization is possible with monovalent ions. Here we present direct AFM evidence of DNA immobilized to mica in the presence of only monovalent ions. Our data depict E. coli plasmid pBR322 adsorbed onto the negatively charged mica both after short (10 min) and long (24 h) incubation periods. These data suggest the need to re‐explore current theories of like‐charge attraction to include the possibility of monovalent interactions. We suggest that this DNA immobilization strategy may offer the potential to image natural processes with limited immobilization forces and hence enable maximum conformational freedom of the immobilized biomolecule.     

Hundred‐Thousand light holes push nanoscopy to go parallel

With the application of the elements of all major super‐resolution techniques including stimulated emission depletion, structure illumination microscopy, and photo‐activated localization microscopy, the incoherent crossed standing‐wave microscopy achieves parallel super‐resolution imaging. Microsc. Res. Tech., 78:8–10, 2015. © 2014 Wiley Periodicals, Inc.     

Bacterial biofilm on successful and failed orthodontic mini‐implants—a scanning electron microscopy study

Mini‐implants have been extensively used in Orthodontics as temporary bone anchorage devices. However, early failure of mini‐implants due to mobility might occur and the colonization of their surfaces by pathogenic bacteria has been referred to as one of the contributing factors. In this study, scanning electron microscopy (SEM) was used to assess the presence of microorganisms adhered to the surface of mini‐implants that failed due to loss of stability. Twelve self‐drilling titanium mini‐implants (1.6 mm diameter × 9.0 mm long) were collected from 12 patients undergoing orthodontic treatment—7 successful and 5 failed mini‐implants. The mean time of permanence in the mouth was 15.8 and 2.4 months for successful and failed mini‐implants, respectively. The devices were placed in the maxilla and/or mandible and removed by the same surgeon and were processed for SEM analysis of the presence of microorganisms on their surfaces (head, transmucosal profile, and body). Extensive bacterial colonization on mini‐implant head and transmucosal profile was observed in all successful and failed mini‐implants. None of the failed mini‐implants exhibited bacteria on its body and only one mini‐implant belonging to the successful (stable) group exhibited bacteria on its body. The results did not suggest a relationship between failure and presence of bacterial colonies on mini‐implant surfaces. Microsc. Res. Tech. 78:1112–1116, 2015. © 2015 Wiley Periodicals, Inc.     

Two‐In‐one sample preparation for plan‐VIew TEM

Transmission electron microscopy (TEM) sample preparation requires special skills, it is time consuming and costly, hence, an increase of the efficiency is of primary importance. This article describes a method that duplicates the yield of the conventional mechanical and ion beam preparation of plan‐view TEM samples. As a modification of the usual procedures, instead of one two different samples are comprised in a single specimen. The two pre‐cut slabs, one from each samples, are embedded side by side in the window of a 3 mm dia Ti disk and the specimen is thinned mechanically and by ion milling until perforation that occurs at the interface of the two different slabs. That, with proper implementation, provides acceptable size thin area for the TEM study of both samples. The suitability of the two‐in‐one method has been confirmed through examples. Microsc. Res. Tech. 78:599–602, 2015. © 2015 Wiley Periodicals, Inc.     

A simple,straightforward correlative live‐cell‐imaging‐structured‐illumination‐microscopy approach for studying organelle dynamics

Most cellular organelles are highly dynamic and continuously undergo membrane fission and fusion to mediate their function. Documenting organelle dynamics under physiological conditions, therefore, requires high temporal resolution of the recording system. Concurrently, these structures are relatively small and determining their substructural organization is often impossible using conventional microscopy. Structured Illumination Microscopy (SIM) is a super resolution technique providing a two‐fold increase in resolution. Importantly, SIM is versatile because it allows the use of any fluorescent dye or protein and, hence, is highly applicable for cell biology. However, similar to other SR techniques, the applicability of SIM to high‐speed live cell imaging is limited. Here we present an easy, straightforward methodology for coupling of high‐speed live cell recordings, using spinning disk (SD) microscopy, with SIM. Using this simple methodology, we are able to track individual mitochondrial membrane fission and fusion events in real time and to determine the network connectivity and substructural organization of the membrane at high resolution. Applying this methodology to other cellular organelles such as, ER, golgi, and cilia will no doubt contribute to our understanding of membrane dynamics in cells. Microsc. Res. Tech. 78:777–783, 2015. © 2015 Wiley Periodicals, Inc.     

Occurrence of fenestral diaphragms and knots in renal glomerular endothelia of diabetic mutant MafA−/−MafK+ mice as revealed in embedment‐free transmission electron microscopy

Using the advantages (high contrast and transparency and efficient 3D viewing) of embedment‐free section transmission electron microscopy (TEM), the occurrence of numerous fenestral diaphragms was clearly shown in 3D en‐face viewing of the renal glomerular capillary endothelium of severe overt diabetes mellitus mice, which were generally MafA‐deficient and simultaneously MafK‐overexpressed specifically in pancreatic β‐cells. This presents another example of nephritis‐induced diaphragmed fenestrae in the renal glomerular endothelium. In addition, knot‐/umbilicus‐like structures discrete from and larger than the central knots of regular diaphragms of fenestrated endothelium were clearly demonstrated to occur randomly in the renal glomerular endothelial fenestrae of mutant mice and wild ones. The knot‐structures were revealed to be protrusions of underlining basement lamina in conventional TEM by section‐tilting observation. Microsc. Res. Tech. 78:207–212, 2015. © 2015 Wiley Periodicals, Inc.     

Detecting changes in arthritic fibroblast‐like synoviocytes using atomic force microscopy

The morphological and quantitative differences between arthritic fibroblast‐like synoviocytes (FLS) and normal FLS were determined as an effective diagnostic tool for rheumatoid arthritis (RA), and confirmed using atomic force microscopy (AFM). Collagen‐induced arthritic (CIA) mice and normal mice were prepared and FLS were isolated by enzymatic digestion from the synovial tissue of sacrificed mice at 5‐week and 8‐week pathogenesis periods. Analysis of cell morphology using AFM revealed that the surface roughness around the nucleus and around the branched cytoplasm was significantly higher in CIA FLS (P < 0.05) than that in normal FLS. In addition, the roughness of two different sites on the arthritic FLS increased with an increase in the duration of pathogenesis. These results strongly suggest that AFM can be widely used as a diagnostic tool in cytopathology to detect the early signs of RA and various others diseases at the intercellular level. Microsc. Res. Tech. 78:982–988, 2015. © 2015 Wiley Periodicals, Inc.