Ultrastructural and three‐dimensional study of post‐LASIK ectasia cornea

INTRODUCTION: Post‐laser in situ keratomileusis (LASIK) corneal ectasia is a serious late postoperative complication. Here, we report the ultrastructural features of the post‐LASIK cornea of two patients. METHODS: Two normal corneas (age 24 and 37 years old) and two post‐LASIK ectaic corneas from two patients (A and B) were studied. The “patient A” (age 27 years) underwent penetrating keratoplasty and “patient B” (age 31 years) underwent deep‐anterior lamellar keratoplasty. The excised corneas were processed for light and electron microscopy. A total of 120 images for three‐dimensional (3D) reconstruction were taken by using the software “Recorder” and using a bottom mounted camera “Quemesa” attached to a JOEL 1400 transmission electron microscope. The 3D images were constructed using “Visual Kai” software. RESULTS: In the post‐LASIK cornea, the hemidesmosomes, the basement membrane, and Bowman”s layer were abnormal. The stromal lamellae were thin and disorganized. The collagen fibrils (CFs) diameter and interfibrillar spacing had decreased. Aggregated microfibrils were present in the Bowman's layer and all parts of the stroma. A large number of microfilaments were present at the detachment end of the flap and residual stroma. The 3D images showed the presence of collagen microfibrils and proteoglycans (PGs) within the CF of the normal and post‐LASIK cornea. The collagen microfibrils and PGs within the CFs had degenerated in the post‐LASIK cornea. CONCLUSION: Collagen microfibrils and PGs within the CFs were degenerated, leading to the degeneration of CFs, followed by the disorganization of lamellae in post‐LASIK cornea. The CFs diameter and interfibrillar spacing decreased. Microsc. Res. Tech. 77:91–98, 2014. © 2013 Wiley Periodicals, Inc.     

Comparison of cyclic fatigue resistance between different NiTi instruments with 4% taper

The aim of this study was to make a comparison between the cyclic fatigue (CF) resistance of F360, twisted files (TF), FlexMaster (FM) and RaCE instruments with 4% taper. A total of 40 instruments were evaluated 8 mm from the tip. A stainless steel block with a simulated canal of 1.5 mm diameter, and a 60° angle of curvature was tested using CF testing. One‐way ANOVA and posthoc Tukey's test (P < 0.05) were used. The F 360 files showed the highest CF resistance while the TF files had greater CF resistance than the FM and RaCE (P < 0.05). There was no significant difference between the FM and RaCE (P > 0.05). F360 instruments with a double S cross‐section had the highest CF resistance among the group. The TF led the NiTi rotary files to be more resistant to fatigue than the FM and RaCE instruments. Microsc. Res. Tech. 79:345–348, 2016. © 2016 Wiley Periodicals, Inc.     

Effects of cutting fluid application on tool wear in machining: Interactions with tool-coatings and tool surface features

Minimal Quantity of Lubricant (MQL) application of cutting fluids (CFs), or near-dry machining, is being proposed as an environmentally and economically viable alternative to conventional flooding under conditions where dry cutting is not feasible. However, several issues related to CF application effects on cutting tool wear need further clarification, especially, the interactions of CF application with tool-coatings and chip-breakers, both of which are widely employed in industrial cutting tools, need further study. This paper presents the results of an experimental study into the effects of different CF application methods on tool wear during machining of AISI 1045 steel using flat-faced and grooved, coated carbide cutting tools. The results provide insight into the mechanisms of tool wear in the presence of CFs, as well as the influence of chip-breaking geometric features, and tool-coating systems, on CF action. The wear mode was observed to be dictated by thermal considerations, rather than by any friction reduction capability of different CF application methods, and forced attempts at achieving lubricating action were negatively affecting tool life under some conditions.     

Cornea and its adaptation to environment and accommodation function in veiled chameleon (Chamaeleo calyptratus): ultrastructure and 3D transmission electron tomography

We investigate the ultrastructural features and 3D electron tomography of chameleon (Chamaeleon calyptratus) which is a native of desert environments of Saudi Arabia. The corneas of the chameleon were fixed in 2.5% glutaraldehyde containing cuprolinic blue in sodium acetate buffer for electron microscopy and tomography, and observed under a JEOL 1400 transmission electron microscope. The thin cornea (21.92 μm) contained 28–30 collagen fibril lamellae. The middle stromal lamellae (from 13 to 19) contained keratocytes with a long cell process and filled with granular material. The CF diameter increased from lamella 1 (30.44 ± 1.03) to Lamella 5 (52.83 ± 2.00) then decreased towards the posterior stoma. The percentage of large CF diameters (55–65 nm) was very high in the lamellae L14 (38.8%) and L15 (85.7%). The mean PGs area of the posterior stroma (448.21 ± 24.84 nm²) was significantly larger than the mean PGs area of the anterior, (309.86 ± 8.2 nm²) and middle stroma 245.94 ± 8.28 nm²). 3D electron tomography showed the distribution of PGs around and over the CF. Variable diameters of CFs in the anterior stroma may provide compact lamellae which may restrict the low wavelength of light. Variable diameters of CFs in the anterior stroma may provide compact lamellae which may restrict the low wavelength of light. This accommodation function is achieved by bending of the cornea. During bending the anterior stroma was stretched and the posterior stroma was compressed due to the presence of small CFs. The middle stroma remained stiff due to the presence of large CFs. Large proteoglycans not only maintain hydration for a longer period of time, but also act as a lubricant to neutralise the shear forces in the anterior and posterior stroma during bending.     

Effect of processing methods for transmission electron microscopy on corneal collagen fibrils diameter and spacing

Introduction: The corneal tissue was processed in fixatives and embedded in resin for transmission electron microscopy to observe the ultrastructure of the collagen fibrils (CFs). The effect of these processing methods on the CF diameter and the interfibrillar spacing was studied. Methods: Four normal human corneal buttons were used for this study. A part of each cornea was fixed in 2.5% glutaraldehyde containing cuprolinic blue in sodium acetate buffer and embedded in spurr's resin (SpurrCB). A second part of each cornea was fixed in 2.5% glutaraldehyde + osmium tetroxide and embedded spurr's resin (SpurrOsm). The third part of each cornea was fixed in paraformaldehyde (4%) and embedded in LR White at 4°C (LRWhite). Ultrathin sections were stained with uranyl acetate and lead citrate. Results: In the tissue, fixed in SpurrCB, the diameter was 38.4 ± 5.9 nm and spacing between CF was 52.5 ± 5.3 nm. In the tissue fixed in SpurrOsm, the diameter was 28.37 ± 5.84 nm and spacing between CF was 45 ± 4.57 nm. In the tissue fixed in LR White, the CF diameter was 24 ± 2.3 nm and spacing between CF was 39.0 ± 4.2 nm. The diameters and interfibrillar spacing of the tissue processed by SpurrCB, SpurrOsm, and LRWhite were significantly different (P < 0.001) from one another. Conclusion: Our study shows that there is a variation in the CF diameter and spacing depending on the method of fixation and embedding resins used. This needs to be considered when comparative studies using different methods are done. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.     

Performance of plastic gear made of carbon fiber reinforced poly-ether-ether-ketone: Part 2

Five kinds of PEEK/CF composites, made by blending poly-ether-ether-ketone (PEEK) with three kinds of PAN type carbon fibers (CFs) and two kinds of pitch type CFs respectively, were injection-molded into gears. Their gear performance such as the load capability and the wear of tooth were evaluated. The wear properties of PEEK/CF gears extremely varied depending on the kinds of CFs, when the same type gears were combined and grease was initially applied. Also, the load capabilities were significantly influenced by the affinity between PEEK and CF. A composite gear reinforced with CF of the highest density indicated the highest load capability irrespective of the test conditions, due to the lowest abrasive property of the CF as well as the excellent affinity between PEEK and CF. Its load capability under a high temperature running condition was found to be superior to that of polyamideimide (PAI) composite gear or polyphenylenesulphide (PPS) composite gear.     

Comparison of two automatic cell‐counting solutions for fluorescent microscopic images

Cell counting in microscopic images is one of the fundamental analysis tools in life sciences, but is usually tedious, time consuming and prone to human error. Several programs for automatic cell counting have been developed so far, but most of them demand additional training or data input from the user. Most of them do not allow the users to online monitor the counting results, either. Therefore, we designed two straightforward, simple‐to‐use cell‐counting programs that also allow users to correct the detection results. In this paper, we present the Cellcounter and Learn 123 programs for automatic and semiautomatic counting of objects in fluorescent microscopic images (cells or cell nuclei) with a user‐friendly interface. Although Cellcounter is based on predefined and fine‐tuned set of filters optimized on sets of chosen experiments, Learn 123 uses an evolutionary algorithm to determine the adapt filter parameters based on a learning set of images. Cellcounter also includes an extension for analysis of overlaying images. The efficiency of both programs was assessed on images of cells stained with different fluorescent dyes by comparing automatically obtained results with results that were manually annotated by an expert. With both programs, the correlation between automatic and manual counting was very high (R² < 0.9), although Cellcounter had some difficulties processing images with no cells or weakly stained cells, where sometimes the background noise was recognized as an object of interest. Nevertheless, the differences between manual and automatic counting were small compared to variations between experimental repeats. Both programs significantly reduced the time required to process the acquired images from hours to minutes. The programs enable consistent, robust, fast and accurate detection of fluorescent objects and can therefore be applied to a range of different applications in different fields of life sciences where fluorescent labelling is used for quantification of various phenomena. Moreover, Cellcounter overlay extension also enables fast analysis of related images that would otherwise require image merging for accurate analysis, whereas Learn 123's evolutionary algorithm can adapt counting parameters to specific sets of images of different experimental settings.     

Nondestructive estimation of growth year in ginseng cultivars using the means of mathematical modeling on the basis of allometry

Growth‐year authentication has extraordinary significance for plant growth, structure and development research, and has a wide range of applications in value assessment of economic crops. Panax ginseng is the most commonly used medicinal plant in Asian countries. The fix number of growth‐year is an important quality evaluation which is difficult to be obtained accurately in current technical conditions. Preliminary authentication theory for growth‐year has been described in previous studies using a short‐lived perennial medicinal plant (Paeonia lactiflora pall.) as the research material. In this research, we focused on the growth‐year estimation in ginseng cultivars, and attempt to explore the age estimation method for vascular plants according to mathematical simulation of the root structure development. Micro data was obtained from 204 individuals of 3 different kinds of ginseng cultivars, which have a series of gradient age and a clear growth record. Outer diameter of the vascular cambium (b) and the radius of cross section (r) were measured with ordinary stereo microscope. We further designed and established two different kinds of authentication model based on the taproot structure development for growth year authentication (P and M = K*X₁^a1X₂^a2). Moreover, the models were applied to identify the growth year of ginseng without damage using Micro‐CT or DEI reconstruction. A potential method, have been recently described, the age of ginseng can be analyzed by telomere length and telomerase activity. However, we found that there are different results indicated in other species. We concluded that microscopic methods perceived currently were provided a more effective means for growth‐year authentication. Microsc. Res. Tech. 79:98–105, 2016. © 2016 The Authors Microscopy Research and Technique Published by Wiley Periodicals, Inc.     

Blind deconvolution of 3D fluorescence microscopy using depth‐variant asymmetric PSF

The 3D wide‐field fluorescence microscopy suffers from depth‐variant asymmetric blur. The depth‐variance and axial asymmetry are due to refractive index mismatch between the immersion and the specimen layer. The radial asymmetry is due to lens imperfections and local refractive index inhomogeneities in the specimen. To obtain the PSF that has these characteristics, there were PSF premeasurement trials. However, they are useless since imaging conditions such as camera position and refractive index of the specimen are changed between the premeasurement and actual imaging. In this article, we focus on removing unknown depth‐variant asymmetric blur in such an optical system under the assumption of refractive index homogeneities in the specimen. We propose finding few parameters in the mathematical PSF model from observed images in which the PSF model has a depth‐variant asymmetric shape. After generating an initial PSF from the analysis of intensities in the observed image, the parameters are estimated based on a maximum likelihood estimator. Using the estimated PSF, we implement an accelerated GEM algorithm for image deconvolution. Deconvolution result shows the superiority of our algorithm in terms of accuracy, which quantitatively evaluated by FWHM, relative contrast, standard deviation values of intensity peaks and FWHM. Microsc. Res. Tech. 79:480–494, 2016. © 2016 Wiley Periodicals, Inc.     

Tensile and Friction Properties of Tin Bronze Matrix Composites Reinforced by Carbon Fibers

Copper-coated carbon fibers and carbon fiber (CF)-reinforced tin bronze matrix composites were prepared by chemical plating and powder metallurgy, respectively. Copper-coated CFs were characterized with field emission scanning electron microscope. The effects of CF volume fraction on the friction properties and tensile properties of the composites were investigated. The results showed that the composites exhibited lower friction coefficient and higher tensile strength than tin bronze 6-6-3 with the chemical composition of Cu-6 wt%Sn-6%Zn-3%Pb. The friction coefficient of the composites decreased with the increasing of the CF volume fraction. The composite with 9 vol.% CFs showed the highest hardness and tensile strength, which were, respectively, about 1.8× and 1.68× higher than those of the tin bronze 6-6-3.     

In situ TEM studies of micron‐sized all‐solid‐state fluoride ion batteries: Preparation,prospects, and challenges

Trustworthy preparation and contacting of micron‐sized batteries is an essential task to enable reliable in situ TEM studies during electrochemical biasing. Some of the challenges and solutions for the preparation of all‐solid‐state batteries for in situ TEM electrochemical studies are discussed using an optimized focused ion beam (FIB) approach. In particular redeposition, resistivity, porosity of the electrodes/electrolyte and leakage current are addressed. Overcoming these challenges, an all‐solid‐state fluoride ion battery has been prepared as a model system for in situ TEM electrochemical biasing studies and first results on a Bi/La_0.9Ba_0.1F_2.9 half‐cell are presented. Microsc. Res. Tech. 79:615–624, 2016. © 2016 Wiley Periodicals, Inc.     

Biotinylated vanadium and chromium sulfide nanoparticles as probes for colocalization of membrane proteins

We report the microemulsion synthesis of vanadium and chromium sulfide nanoparticles (NPs) and their biological application as nanoprobes for colocalization of membrane proteins. Spherical V₂S₃ and Cr₂S₃ NPs were prepared in reverse microemulsion droplets, as nanoreactors, obtained by the surfactant sodium bis(2‐ethylhexyl) sulfosuccinate (AOT) in nonpolar organic phase (heptane). Electron microscopic data indicated that the size distribution of the nanoparticles was uniform with an average diameter between 3 ÷ 5 nm. The prepared hydrophobic nanocrystals were transferred in aqueous phase by surface cap exchange of AOT with biotin‐dihydrolipoic ligands. This substitution allows the nanoparticles solubility in aqueous solutions and confer their bioactivity. In addition, we report the conjugation procedure between α‐Lipoic acid (LA) and biotin (abbreviated as biotin‐LA). The biotin‐LA structure was characterized by 1D and 2D NMR spectroscopy. The biotinylated vanadium and chromium sulfide nanoparticles were tested as probes for colocalization of glutamate receptors on sodium‐dodecyl‐sulfate‐digested replica prepared from rat hippocampus. The method suggests their high labeling efficiency for study of membrane biological macromolecules. Microsc. Res. Tech. 79:799–805, 2016. © 2016 Wiley Periodicals, Inc.     

Making the practically impossible “Merely difficult”—Cryogenic FIB lift‐out for “Damage free” soft matter imaging

The preparation of thinned lamellae from bulk samples for transmission electron microscopy (TEM) analysis has been possible in the focussed ion beam scanning electron microscope (FIB‐SEM) for over 20 years via the in situ lift‐out method. Lift‐out offers a fast and site specific preparation method for TEM analysis, typically in the field of materials science. More recently it has been applied to a low‐water content biological sample (Rubino 2012). This work presents the successful lift‐out of high‐water content lamellae, under cryogenic conditions (cryo‐FIB lift‐out) and using a nanomanipulator retaining its full range of motion, which are advances on the work previously done by Rubino (2012). Strategies are explored for maintaining cryogenic conditions, grid attachment using cryo‐condensation of water and protection of the lamella when transferring to the TEM. Microsc. Res. Tech. 79:298–303, 2016. © 2016 Wiley Periodicals, Inc.     

Integration of a high‐NA light microscope in a scanning electron microscope

We present an integrated light‐electron microscope in which an inverted high‐NA objective lens is positioned inside a scanning electron microscope (SEM). The SEM objective lens and the light objective lens have a common axis and focal plane, allowing high‐resolution optical microscopy and scanning electron microscopy on the same area of a sample simultaneously. Components for light illumination and detection can be mounted outside the vacuum, enabling flexibility in the construction of the light microscope. The light objective lens can be positioned underneath the SEM objective lens during operation for sub‐10 μm alignment of the fields of view of the light and electron microscopes. We demonstrate in situ epifluorescence microscopy in the SEM with a numerical aperture of 1.4 using vacuum‐compatible immersion oil. For a 40‐nm‐diameter fluorescent polymer nanoparticle, an intensity profile with a FWHM of 380 nm is measured whereas the SEM performance is uncompromised. The integrated instrument may offer new possibilities for correlative light and electron microscopy in the life sciences as well as in physics and chemistry.     

Strong hydrophobic interaction between graphene oxide and supported lipid bilayers revealed by AFM

Understanding the interaction between graphene oxide (GO) and lipid membranes is of great importance for its various applications in biotechnology. Here, we investigated the interaction between GO and charged supported lipid bilayers (SLBs) by in situ atomic force microscope (AFM) imaging. It was found that GO could peel off a single layer of positively charged SLBs and deposited on the hydrophobic part of the remaining sublayer. Then free lipid molecules would assemble on GO surface and formed 1.5 bilayers in a lipid‐GO‐lipid manner. For negatively charged lipid bilayers, however, GO deposited to the SLBs only when its concentration was very high. These results indicate that, in addition to electrostatic interaction, the hydrophobic interaction plays an important role when GO sheets deposit onto the charged lipid bilayers, and should be helpful to understand possible cytotoxicity and antibiosis of graphene‐related nanomaterials. Microsc. Res. Tech. 79:721–726, 2016. © 2016 Wiley Periodicals, Inc.     

Atomic force microscopy study of living diatoms in ambient conditions

We present the first in vivo study of diatoms using atomic force microscopy (AFM). Three chain‐forming, benthic freshwater species –Eunotia sudetica, Navicula seminulum and a yet unidentified species – are directly imaged while growing on glass slides. Using the AFM, we imaged the topography of the diatom frustules at the nanometre range scale and we determined the thickness of the organic case enveloping the siliceous skeleton of the cell (10 nm). Imaging proved to be stable for several hours, thereby offering the possibility to study long‐term dynamic changes, such as biomineralization or cell movement, as they occur. We also focused on the natural adhesives produced by these unicellular organisms to adhere to other cells or the substratum. Most man‐made adhesives fail in wet conditions, owing to chemical modification of the adhesive or its substrate. Diatoms produce adhesives that are extremely strong and robust both in fresh‐ and in seawater environments. Our phase‐imaging and force‐pulling experiments reveal the characteristics of these natural adhesives that might be of use in designing man‐made analogues that function in wet environments. Engineering stable underwater adhesives currently poses a major technical challenge.     

Performance of scientific cameras with different sensor types in measuring dynamic processes in fluorescence microscopy

The plethora of available scientific cameras of different types challenges the biologically oriented experimenter when picking the appropriate camera for his experiment. In this study, we chose to investigate camera performances in a typical nonsingle molecule situation in life sciences, that is, quantitative measurements of fluorescence intensity changes from video data with typically skewed intensity distributions. Here, intensity profile dynamics of pH‐sensors upon triggered changes of pH‐environments in living cells served as a model system. The following camera types were tested: sCMOS, CCD (scientific and nonscientific) and EM‐CCD (back‐ and front‐illuminated). We found that although the EM‐CCD cameras achieved the best absolute spatial SNR (signal‐to‐noise ratio) values, the sCMOS was at least of equal performance when the spatial SNR was related to the effective dynamic range, and it was superior in terms of temporal SNR. In the measurements of triggered intensity changes, the sCMOS camera had the advantage that it used the smallest fraction of its dynamic range when depicting intensity changes, and thus featured the best SNR at full usage of its dynamic range. Microsc. Res. Tech. 76:835–843, 2013. © 2013 Wiley Periodicals, Inc.     

Immunohistochemical and ultrastructural evidence of functional organization along the Corydoras paleatus intestine

The Neotropical catfish, Corydoras paleatus (Callichthyidae) is a facultative air‐breathing teleost that makes use of the caudal portion of the intestine as an accessory air‐breathing organ. This portion is highly modified, being well vascularized with capillaries between epithelial cells, which makes it well suited for gas exchange. Instead, the cranial portion is a digestion and absorption site, as it has a typical intestinal epithelium with columnar cells arranged in a single row, villi and less vascularized tunica mucosa. Therefore, the intestine was studied by light and electron microscopy to assess differences between the cranial, middle and caudal portions. To characterize the potential for cell proliferation of this organ, we used anti‐proliferating cell nuclear antigen antibody and anti‐Na⁺K⁺‐ATPase monoclonal antibody to detect the presence of Na⁺/K⁺ pump. In C. paleatus it was observed that cell dynamics showed a decreasing gradient of proliferation in cranio‐caudal direction. Also, the intestine of this catfish is an important organ in ionoregulation: the basolateral Na⁺/K⁺ pump may have an active role, transporting Na⁺ out of the cell while helping to maintain the repose potential and to regulate cellular volume. Microsc. Res. Tech. 79:140–148, 2016. © 2016 Wiley Periodicals, Inc.     

A MEMS‐based heating holder for the direct imaging of simultaneous in‐situ heating and biasing experiments in scanning/transmission electron microscopes

The introduction of scanning/transmission electron microscopes (S/TEM) with sub‐Angstrom resolution as well as fast and sensitive detection solutions support direct observation of dynamic phenomena in‐situ at the atomic scale. Thereby, in‐situ specimen holders play a crucial role: accurate control of the applied in‐situ stimulus on the nanostructure combined with the overall system stability to assure atomic resolution are paramount for a successful in‐situ S/TEM experiment. For those reasons, MEMS‐based TEM sample holders are becoming one of the preferred choices, also enabling a high precision in measurements of the in‐situ parameter for more reproducible data. A newly developed MEMS‐based microheater is presented in combination with the new NanoEx?‐i/v TEM sample holder. The concept is built on a four‐point probe temperature measurement approach allowing active, accurate local temperature control as well as calorimetry. In this paper, it is shown that it provides high temperature stability up to 1,300°C with a peak temperature of 1,500°C (also working accurately in gaseous environments), high temperature measurement accuracy (<4%) and uniform temperature distribution over the heated specimen area (<1%), enabling not only in‐situ S/TEM imaging experiments, but also elemental mapping at elevated temperatures using energy‐dispersive X‐ray spectroscopy (EDS). Moreover, it has the unique capability to enable simultaneous heating and biasing experiments. Microsc. Res. Tech. 79:239–250, 2016. © 2016 Wiley Periodicals, Inc.     

Intracellular imaging of Qdots‐labeled DNA in cyanobacteria

In this contribution, they have attempted to develop a labeling technique for in vivo imaging of functionally active plasmid DNA in cyanobacterial cells through its decoration with semiconductor quantum dots (Qdots) as fluorescent nanoprobes. For that purpose biotinylated plasmid slr2060 DNA was conjugated with Qdots‐streptavidine. The intact DNA was visualized in a single green color by light microscopy. These Qdots‐DNA conjugates were capable of expressing the acyltransferase enzyme. Qdots‐DNA conjugates and confocal microscope imaging technique were adopted to visualize the gene transport across the membrane of the live cyanobacteria cell in real time. Long‐term kinetic study enabled to reveal the steps of extracellular and intracellular microenvironment for plasmid transportation into the live cell. To confirm these processes a confocal microscope and indicator plate assay test were applied in tandem. In this contribution, Qdots‐labeled plasmid DNA was utilized for the first time for long‐term intracellular imaging studies in cyanobacteria species PCC6803. The results showed that the Qdots‐labeled plasmid DNA detection could be used as a powerful labeling technique for visualization of exogenous DNA entry and tracking into living cells by confocal microscopy. Microsc. Res. Tech. 79:447–452, 2016. © 2016 Wiley Periodicals, Inc.