Epoxy Sections Research Articles

Fundus Autofluorescence Variation in Geographic Atrophy of Age-Related Macular Degeneration: A Clinicopathologic Correlation.

The purpose of this study was to develop ground-truth histology about contributors to variable fundus autofluorescence (FAF) signal and thus inform patient selection for treating geographic atrophy (GA) in age-related macular degeneration (AMD). One woman with bilateral multifocal GA, foveal sparing, and thick choroids underwent 535 to 580 nm excitation FAF in 6 clinic visits (11 to 6 years before death). The left eye was preserved 5 hours after death. Eye-tracked ex vivo imaging aligned sub-micrometer epoxy resin sections (n=140, 60 µm apart) with clinic data. Light microscopic morphology corresponding to FAF features assessed included drusen-driven atrophy, persistent hyperautofluorescence (hyperFAF) islands and peninsulas within atrophy, and hyperFAF and hypoautofluorescence (hypoFAF) inner junctional zone (IJZ) and outer junctional zone (OJZ) relative to descent of external limiting membrane (ELM). Atrophy growth rate was calculated. HypoFAF atrophic spots appeared in association with drusen, and then expanded and coalesced. Over drusen (n=45, all calcified), RPE was continuous and thin, photoreceptors were short or absent, and initially intact ELM descended where RPE was absent. In persistent hyperFAF within atrophy and in the OJZ, the RPE was continuous and dysmorphic, photoreceptors were present and short, and BLamD was thick. In the IJZ, mottled FAF corresponded to dissociated RPE atop persistent BLamD. Overall linear growth rate (0.198 mm/ year) typified multifocal GA. FAF in GA is locally multifactorial, with photoreceptor shortening potentially promoting hyperFAF by increasing incoming excitation light available to RPE fluorophores. RPE dysmorphia may lead to either longer or shorter pathlength for excitation light. At both atrophy initiation and expansion Müller glia are major participants.

Morphofunctional assessment of muscles of the lower leg and foot after autoneuroplasty of resection defect of tibial portion of the sciatic nerve and single intraoperative electrical neurostimulation in adult rats

INTRODUCTION: There are no data in the literature on the effect of a single intraoperative electrical stimulation (IES) on the condition of the muscles of the lower leg and foot in the long-term period after autoplasty of the sciatic nerve in adult rats. AIM: To study morphofunctional characteristics of muscles of the lower leg and foot after autoneuroplasty of a resection defect of the tibial portion of the sciatic nerve and a single IES in adult rats. MATERIALS AND METHODS: The experiment was performed on 30 Wistar rats who underwent autoneuroplasty (AN) after resection of the tibial portion of the sciatic nerve. Fourteen rats underwent a 40-munite IES session (AN + IES series). In 16 rats IES was not conducted (AN series). At 4 and 6 months after the operation, the tibial nerve function index (TFI) was calculated by analyzing rats’ paw traces on a walking track. At the same time, light microscopy and histomorphometry of paraffin and epoxy sections of gastrocnemius and plantar interosseous muscles were performed. A conventional control was muscles of intact limbs. RESULTS: Atrophy and endomysial fibrosis were less expressed in the gastrocnemius of the AN + IES series in comparison with AN series, the effect was mediated by enhancement of vascularization. In plantar interosseous muscles at 4 months after the operation, the volume density of blood vessels in the AN + IES series was 7.35 (5.49; 8.69), which was greater than in the AN series — 3.43 (2.02; 5.59), р = 0.0196. Diameters of muscle fibers and volume density of endomysium were comparable. At 6 months after the operation, endomysial fibrosis progressed in both series, but in the AN + IES series, myopathically altered muscle fibers were less common. After 6-month observation, TFI increased (-47.95) in the AN + IES series and became higher (p = 0.0339) than in the AN series, in which TFI became even lower (-93.64) than it was after 4 months (-81.95) of the experiment. CONCLUSION: A single IES permits to reduce the denervation alterations in the gastrocnemius and plantar interosseous muscles conditioned by damage to the nerve and maturation, and also to improve the tibial nerve function index in the long term after autoneuroplasty.

Microvessels’ Atheroscleroses Study and Evaluation of Some Patients with Coronary Artery Disease at Open Heart Surgery in Armenia

Ischemic heart disease is a rather insidious condition with a high mortality rate, especially in developing countries, which also affects Armenia. Therefore, the issue of the most effective treatment for this disease is particularly pressing. This will be facilitated by more thorough research on atherosclerosis in arterioles. The ability to visualize and assess pathological vascular growth on a 5-point scale is provided by one of the modern research methods using semi-thin epoxy sections stained with Azure II. The bioptates of the right atrial auricles of 4 patients with coronary artery disease were taken during coronary artery bypass grafting. The epoxy slices were obtained from material treated by the method of transmission electron microscopy and embedded in epoxy resin, with further staining by Azur II, examined under light optical microscopy. The obtained images of the morphological picture allowed for the identification of atherosclerotic changes in the arterioles of the myocardium of the right atrial appendage in patients, which exhibited pronounced growth resembling intussusception and were accordingly assessed on a 5-point scale for pathological growth of the microcirculatory bed was rated at 3-4 points for one patient and 3 points for three patients. Provided 1-2 images could help in the analysis of atherosclerotic damages and the identification of the direction of the pathological process in each patient case. This will assist both cardiac surgeons and cardiologists in selecting the appropriate medication therapy during the postoperative and separate periods.

Imaging Histology Correlations of Intraretinal Fluid in Neovascular Age-Related Macular Degeneration.

Fluid presence and dynamism is central to the diagnosis and management of neovascular age-related macular degeneration. On optical coherence tomography (OCT), some hyporeflective spaces arise through vascular permeability (exudation) and others arise through degeneration (transudation). Herein we determined whether the histological appearance of fluid manifested this heterogeneity. Two eyes of a White woman in her 90s with anti-vascular endothelial growth factor treated bilateral type 3 neovascularization secondary to age-related macular degeneration were osmicated, prepared for submicrometer epoxy resin sections, and correlated to eye-tracked spectral domain OCT. Examples of intraretinal tissue fluid were sought among similarly prepared donor eyes with fibrovascular scars, in a web-based age-related macular degeneration histopathology resource. Fluid stain intensity was quantified in reference to Bruch's membrane and the empty glass slide. Exudative fluid by OCT was slightly reflective and dynamically responded to anti-vascular endothelial growth factor. On histology, this fluid stained moderately, possessed a smooth and homogenous texture, and contained blood cells and fibrin. Nonexudative fluid in degenerative cysts and in outer retinal tubulation was minimally reflective on OCT and did not respond to anti-vascular endothelial growth factor. By histology, this fluid stained lightly, possessed a finely granular texture, and contained mainly tissue debris. Quantification supported the qualitative impressions of fluid stain density. Cells containing retinal pigment epithelium organelles localized to both fluid types. High-resolution histology of osmicated tissue can distinguish between exudative and nonexudative fluid, some of which is transudative. OCT and histological features of different fluid types can inform clinical decision-making and assist in the interpretation of newly available automated fluid detection algorithms.

Effects of the strain gradients on the band structures of the elastic waves propagating in 1D phononic crystals: An analytical approach

In this paper, an analytical method is proposed for the analysis of the effects of the strain gradients on the frequency band structures and band-gaps of the elastic waves propagating in one-dimensional (1D) nano-sized phononic crystals (PCs). The considered 1D nano-sized PCs are made of periodic unit-cells containing a metal section and an epoxy section. The strain gradients are taken into account to obtain the governing equations by using the strain energy, the kinetic energy and the Hamilton's principle. An analytical solution based on the transfer matrix method is derived to compute the frequency dispersion curves or band structures of the elastic waves. The continuity conditions on the interface between the neighboring sections and the periodic boundary conditions for the unit-cell are satisfied using the proposed analytical method. Two band-gap characteristics, namely the starting frequency and the width of each band-gap, are defined and investigated. The effects of the small-scale parameters in the strain-gradient theory on the band structures and the band-gap characteristics are analyzed and discussed in detail. It is demonstrated that the strain gradients may significantly influence the frequency band structures and the band-gap characteristics of the elastic waves propagating in 1D nano-sized PCs.

Prediction of Mechanical Behavior of Epoxy Polymer Using Artificial Neural Networks (ANN) And response Surface Methodology (RSM)

The aim of this study is to analyze the effect of different geometries and sections on the mechanical properties of epoxy specimens. Five tensile tests were carried out on three types of series. The experimental results obtained were 1812.21 MPa, 3.90% and 41.91 MPa for intact specimens, 1450.41 MPa, 2.16% and 21.28 MPa for specimens with hole and 750.77 MPa, 2.77% and 11.89 MPa for specimens with elliptical -notched for Young's modulus, strain and stress respectively. In addition, the experimental results indicated that the mechanical properties of both (Young's modulus value and stress value) were higher in an intact specimen. Afterwards, the nonlinear functional relationship of input parameters between epoxy sample geometries and sections was established using the response surface model (RSM) and the artificial neural network (ANN) to predict the output parameters of mechanical properties (Young's modulus and stress). In addition, the design of experiment was developed by the Analysis of the Application of Variance (ANOVA). The results showed the superiority of the ANN model over the RSM model, where the correlation coefficient values for the model datasets exceed ANN (R2 = 0.984 for Young's modulus and R2 = 0.981 for the constraint).

Sural Nerve Biopsy Assessment by Individual Histologic Preparations: An Observational Cohort Study of 100 Consecutive Cases (P11-8.001)

<h3>Objective:</h3> To study sural nerve biopsy utility based on different histopathologic techniques. <h3>Background:</h3> Nerve biopsies in select patients assist neuropathy diagnosis. Systematic study of their value to inform and alter treatment recommendations quantifying the value of separate histologic preparations is lacking in evidence-based practice. <h3>Design/Methods:</h3> Consecutive sural nerve biopsies (50 internal and 50 external referrals) were reviewed. Standard histological preparations plus graded teased nerve fibres (GTNF), immunohistochemistry, and epoxy-semithin morphometric analysis were studied. Nerve fibre and interstitial abnormalities were scored for each preparation by three examiners masked to case identification. Multivariate modeling was used to inform on the best combination of tests vs a gold standard of the full biopsy report plus morphometric analysis. Resulting clinicopathological diagnosis and treatment recommendations were reviewed. <h3>Results:</h3> Paraffin-stained sections best recognized interstitial abnormalities: Epineurial inflammation (n=59); vasculitis with vessel wall destruction (n=14); amyloidosis (n=2); and noncaseating granuloma (n=1). Vasculitic neuropathy associated with GTNF axonal degeneration (79%) with OR 3.8, 95%CI [1.001, 14.7], p=0.04, not significantly seen with the other preparations. Teased fibre abnormalities correlated with clinicopathologic diagnosis in demyelinated fibers in chronic inflammatory demyelinating polyradiculoneuropathy, 80% (8/10); amyloidosis, 50% (1/2); adult-onset polyglucosan disease 100% (1/1). GTNF and paraffin stains significantly correlated with fibre density determined by morphometric analysis (GTNF: OR 9.9, p&lt;0.0001, paraffin: OR 3.8, p=0.03), not significant with semithin epoxy: OR 1.1, p=0.90, or immunohistochemistry: OR 2.4, p=0.18). GTNF combined with paraffin sections had the highest accuracy for predicting clinicopathologic diagnosis and fibre density with 0.86 C-stat prediction versus morphometric analysis. Among internal cases sural biopsy aided clinicopathologic diagnosis: immunotherapy initiation (44%); reduced immunotherapy (18%); and escalated immunotherapy (8%). <h3>Conclusions:</h3> Sural nerve biopsy have high diagnostic utility frequently altering treatment recommendations in select patients. Paraffin stains combined with GTNF provide highest diagnostic utility, confidence, inter-rater reliability, and accuracy for diagnosis. Immunostains and epoxy sections have focused utility. <b>Disclosure:</b> Dr. Soontrapa has nothing to disclose. Dr. Dyck has nothing to disclose. Dr. Dyck has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Akcea/Ionis. JaNean Engelstad has nothing to disclose. An immediate family member of Jenny Davies has received personal compensation for serving as an employee of IBM. An immediate family member of Jenny Davies has stock in IBM. An immediate family member of Jenny Davies has stock in Kyndryl. Dr. Shelly has nothing to disclose. Mr. Harmsen has nothing to disclose. Dr. Mandrekar has nothing to disclose. Robert Spinner has received personal compensation for serving as an employee of Mayo Clinic. Cris M. Ida, 4790 has nothing to disclose. Dr. Klein has a non-compensated relationship as a Klein with Neurology Journal that is relevant to AAN interests or activities.

A lower motor neuron disease in takahē (Porphyrio hochstetteri) is an endoplasmic reticulum storage disease

Aims: To investigate the pathogenesis of a disease in takahē with intracytoplasmic inclusion bodies in lower motor neurons. Methods: Four cases aged between 5 and 12 years, from three different wildlife sanctuaries in New Zealand were examined. Of these, only one had signs of spinal dysfunction in the form of paresis. Stained paraffin sections of tissues were examined by light microscopy and immunostained sections of the ventral horn of the spinal cord by confocal microscopy. Epoxy resin sections of the bird with spinal dysfunction were examined by electron microscopy. Results: Two types of inclusion bodies were noted, but only in motor neurons of the ventral spinal cord and brain stem. These were large globoid eosinophilic bodies up to 5 µm in diameter, and yellow/brown granular inclusions mostly at the pole of the cell. The globoid bodies stained with Luxol fast blue but not with periodic acid Schiff (PAS), or Sudan black. The granular inclusions stained with Luxol fast blue, PAS and Sudan black. Both bodies were slightly autofluorescent. On electron microscopy the globoid bodies had an even electron-dense texture and were bound by a membrane. Beneath the membrane were large numbers of small intraluminal vesicles. The smaller granular bodies were more heterogeneous, irregularly rounded and membrane-bound accumulations of granular electron-dense material, often with electron-lucent vacuoles. Others were more vesicular but contained varying amounts of electron-dense material. The large globoid bodies did not immunostain for lysosomal markers lysosomal associated protein 1 (LAMP1) or cathepsin D so they were not lysosomal. The small granular bodies stained for cathepsin D by a chromogenic method. A kindred matrix analysis showed two cases to be as closely related as first cousins, and another case was almost as closely related to one of them, but the fourth bird was unrelated to any other. Conclusions: It was concluded that this was an endoplasmic reticulum storage disease due to a specific protein misfolding within endoplasmic reticulum. It was rationalised that the two types of inclusions reflected the same aetiology, but that misfolded protein in the smaller granular bodies had entered the lysosomal system via endoplasmic reticulum autophagy. Although the cause was unclear, it most likely had a genetic aetiology or predisposition and, as such, has clinical relevance.

Band structure analysis of Green-Naghdi thermoelastic wave propagation in a GPLs/CNTs-reinforced metamaterial with energy dissipation

In this paper, the band structure analysis of the thermoelastic wave propagation in a phononic crystal (PC), which it is reinforced by graphene platelets (GPLs) and carbon nanotubes (CNTs), is presented. The reinforced PC beam is made of periodic unit-cells including a metal section and a GPLs/CNTs-reinforced epoxy section. The Green-Naghdi theory of the generalized coupled thermoelasticity with energy dissipation and a new modified micromechanical model to predict the mechanical properties of the reinforced section are employed in the analysis. An analytical solution and the transfer matrix method are adopted to compute the band structures. The frequency band-gaps are obtained and the effects of the GPLs and CNTs volume fractions on the width and location of the band-gaps are analyzed and discussed in details. The results show that the GPLs influence the frequency band-gaps and the band structures of the thermoelastic wave propagation in the reinforced PC beam much more remarkably than the CNTs. The proposed analytical solution method, the modified micromechanical model and the presented results can be employed for designing the reinforced PC structures by the GPLs, CNTs or both of them and also for tuning the band-gaps in the future researches.

Rediscover Potassium Permanganate as a Stain for Basic Proteins on Ultrathin Sections at Transmission Electron Microscopy.

Potassium permanganate solution has been used both as a fixative and as a staining for ultrathin sections at transmission electron microscopy, due to its ability to provide good contrast of different tissue components. Subsequently, it has been forgotten due to disadvantages such as conspicuous formation of precipitates and fragility of the tissue sections treated with this dye when placed under the electron beam. Here we demonstrate that the observed granularity of the sections is not related to the formation of non-specific precipitates, but rather to basic proteins such as chromatin proteins closely associated with DNA and ribosomal particles which are intensely stained. This results in a marked contrast of the nuclei, in particular of the heterochromatin areas, the granular component of the nucleoli, and the rough endoplasmic reticulum, that are rich in these protein complexes. We also show how the embedding in LR white acrylic resin can preserve a good morphology and be less sensitive to the treatment with potassium permanganate than the epoxy resin sections, also allowing to perform immunocytochemistry. The fragility of the epoxy resin sections can be partially improved by using formvar-coated grids.

Tension and Torsion Sensing With a Double-Taper Mach-Zehnder Interferometer

A novel setup for a Double-Taper Mach-Zehnder Interferometer (DTMZI) is presented to measure axial strain and torsion. An epoxy section is added between the two tapers as a fixture to ensure torsion sensing. The modal filtering effect of the epoxy upon the interferometry response is experimentally verified. The transmission spectrum of DTMZI under strain and torsion is modelled and further analyzed through the differential accumulated phase between the beating modes.The experimental spectral observation confirms our estimate. The relationships between the spectral shifts in wavelength and applied displacements are obtained. The sensor shows a very high sensitivity against the applied strain and twists.

Fundus Autofluorescence in Neovascular Age-Related Macular Degeneration: A Clinicopathologic Correlation Relevant to Macular Atrophy

Macular atrophy (MA) of retinal pigment epithelium (RPE) and photoreceptors leads to vision loss in neovascular age-related macular degeneration (nAMD) despite successful treatment with antiangiogenic agents. To enhance understanding of MA, fortify the cellular basis of fundus autofluorescence (FAF) imaging, and inform management of nAMD, we performed histologic analysis of an eye with multimodal clinical imaging and apparent prior exudation due to nAMD. Case study and clinicopathologic correlation. A White woman in whom age-related macular degeneration (AMD) findings of inactive subretinal fibrosis (right eye) were followed for 9 years using FAF and OCT, with no detectable subretinal fluid or other recurrent exudation and no intravitreal injections before her death at age 90 years. The right eye was preserved 6.25 hours after death, postfixed in osmium tannic acid paraphenylenediamine, and prepared for submicrometer epoxy resin sections (n = 115), with 19 matched to clinical OCT B-scans. Light microscopic morphology of a hyperautofluorescent (hyperFAF) area due to prior exudation ("floodplain" hyperFAF), hypoautofluorescent (hypoFAF) spots of MA, and areas of unremarkable FAF. Floodplain hyperFAF was visible throughout the 9 years of follow-up, with several hypoFAF atrophic spots expanding within it over time. The hyperFAF pattern corresponded to outer retinal atrophy (ORA) on OCT and photoreceptor loss over dysmorphic yet continuous RPE in histology. The hypoFAF spots inside the floodplain corresponded to complete RPE and outer retinal atrophy (cRORA) on OCT and loss of both photoreceptors and RPE in histology. In contrast, areas of unremarkable FAF showed continuous RPE accompanied by full-length photoreceptors and a thick outer nuclear layer. This direct clinicopathologic correlation for FAF imaging is the first for nAMD. Fundus autofluorescence is a projection image that involves optical signal modulation by photoreceptors as well as emission signal sources in RPE. Hyperautofluorescence due to an exudative floodplain signifies loss of photoreceptors over continuous RPE. Hypoautofluorescence in MA signifies loss of both cell layers. For maximal value, fundus autofluorescence imaging should be interpreted with the multilayer perspective provided by OCT. Prevention of exudation in nAMD may preserve photoreceptors.

Morphological condition of the pulp of intact and affected by caries third molars

To date, there is a theory that increased resistance to caries is observed in the teeth, which for any reason underwent destructive changes in the pulp. That is why there is a need to study the impact of pulp vitality on the development of the carious process. The aim of the research was to study the microscopic structure of epoxy sections of intact and carious third molars. We studied 4 intact and 6 carious third molars. For this purpose, specimens were made taking into account the free penetration of the fixative solution into the pulp. To this end, immediately after the tooth was removed, we cut off its roots almost near the crown, preserving the integrity of the latter. The method relied on the impregnation of specimens with epoxy resin, according to the method of epoxy plastination of tooth specimens, developed at the Department of Human Anatomy of Ukrainian Medical Stomatological Academy. The epoxy blocks were cut with a disk into two halves until the hard tissues of the tooth crown were exposed together with the pulp. We found that the hard tissues (dentin and enamel coating) of intact third molars did not have any structural defects. However, their pulp chamber contained mainly an amorphous substance, devoid of any typical pulp tissue structures. That is, the pulp was in a state of complete devolution. Quite the opposite presentation was observed in specimens of carious teeth. We found that their pulp chamber contained quite noticeable tissue structures typical of the dental pulp. It is interesting that in the subodontoblastic layer, in front of the carious alteration of the enamel, there was compaction of the pulp, which may be due to infiltration of perivascular connective tissue by immunocompetent cells. It was found that on the border with carious destruction of enamel, there was a compacted spot of altered dentin, whose matrix was intensely pigmented in brown colour, due to the accumulation of melanin on the dentino-enamel junction. Its excessive formation is associated with the destruction of protein-carbohydrate complexes of organic matter in the deep layers of dentin. We found that the pulp compaction and the focus of carious alteration of the enamel are projectively connected by a radial cord of altered dentin, known in the literature as “dead tracts”. Hence, there is reason to believe that the identified changes indicate a latent form of caries, with a pulpogenic mechanism of development. Thus, it can be argued that the teeth, which for any reason underwent degenerative changes in the pulp, are not prone to carious lesions, whereas in carious teeth, the pulp is active and involved in the pathogenesis of the carious process.

Stages of Drusen-Associated Atrophy in Age-Related Macular Degeneration Visible via Histologically Validated Fundus Autofluorescence

To determine histologic correlates for stages of drusen-associated atrophy observed with fundus autofluorescence (FAF) and color fundus photography (CFP), of eyes with advanced age-related macular degeneration (AMD). Case study and clinicopathologic correlation. A white woman with AMD findings of inactive subretinal fibrosis (right eye) and untreated nonexudative type 1 macular neovascularization (left eye) was followed for 9 years before death at 90 years of age. Eyes preserved 6.25 hours after death were postfixed in osmium tannic acid paraphenylenediamine and were prepared for submicrometer epoxy resin sections (115 and 90 from the right and left eye, respectively), with 19 aligned to clinical B-scans. Drusen visible by CFP at the last visit were assigned to 4 stages ofFAF: stage 1, isoautofluorescence; stage 2, mildly uniform hyperautofluorescence; stage 3, a ring of hyperautofluorescence around a center of the hypoautofluorescence; and stage 4, uniform hypoautofluorescence. Light microscopic morphologic features at known FAF stages, including druse size, druse contents, and changes in overlying retinal pigment epithelium (RPE), photoreceptors, and external limiting membrane (ELM). Histologic examination of 166 drusen demonstrated that stage 1 isoautofluorescent drusen were visible on CFP. Hyperautofluorescence in stage 2 corresponded to short photoreceptors and complete coverage by RPE. Hypoautofluorescence in stages 3 and 4 corresponded to different extents of RPE atrophy (RPE gap and no RPE, respectively). Of stage 4 drusen, 67% showed no outer nuclear layer (ONL) and an undetectable ELM. Stage 4 included a high proportion of refractile drusen (82%) with many calcific nodules, visible on CFP. We present the first direct clinicopathologic correlation for FAF imaging of drusen-associated atrophy. Our data support 4 FAF stages of drusen-associated atrophy. Stage 2 is the earliest detected stage in which loss of screening by photoreceptor photopigment contributes to uniform hyperautofluorescence. Stages 3 and 4 comport with incomplete RPE and outer retinal atrophy as defined by the Classification of Atrophy Meetings group. Loss of RPE, ONL, and ELM in stage 4 indicates that atrophy can begin over individual drusen. Findings will help the identification of new therapeutic approaches and clinical study end points.

Investigation on the effect of GMA-graft structure in SAN-g-GMA-coSt on inter-phase behavior, thermal stability, and mechanical properties of PC/SAN/GO nanocomposites

In this study, the effect of simultaneous presence of graphene oxide (GO) nanoparticles and styrene-assisted glycidyl-methacrylate-grafted SAN copolymer (SAN-g-GMA-coSt), as a compatibilizer, on inter-phase behavior, microstructure, thermal, and mechanical properties of PC/SAN blends was investigated. First, the structure of GMA-grafts on the surface of the SAN copolymer was adjusted to improve the compatibility of the blend components. The role that processing conditions play in grafting efficiency was comprehensively studied by FTIR and H-NMR techniques. The results indicated that the optimized SAN-g-GMA-coSt contained grafted sequences consist of 5 GMA molecules dispersed in every 387 carbons of the straight SAN chains. This structure of GMA-grafts reduced phase separations of the blend components and the size of droplets. GO localization in the system, controlled by two-step mixing method, was studied theoretically and the prediction was confirmed by the experimental results. The DSC and DMTA thermograms illustrated that the inter-phase region expanded probably by interactions between functional groups on the surface of GO and the epoxy section of GMA. In addition, thermogravimetric and mechanical results indicated that incorporation of nanoparticles in the presence of SAN-g-GMA-coSt increased the residual mass (char yield) at 600 C and tensile strength of nanocomposites by 55% and 37%, respectively.

Nonexudative Macular Neovascularization Supporting Outer Retina in Age-Related Macular Degeneration: A Clinicopathologic Correlation

Type 1 macular neovascularization (MNV) secondary to age-related macular degeneration (AMD) may sustain hypoxic and micronutrient-insufficient outer retinal cells compensatorily. We explored this hypothesis via histologic analysis of an eye with a shallow irregular retinal pigment epithelial elevation (SIRE) on OCT and good vision. Case study and clinicopathologic correlation. A white woman with untreated nonexudative neovascular AMD and 20/30 visual acuity (left eye) and neovascular AMD (right eye), with 9 years' multimodal imaging before dying at 90 years of age. The left eye was preserved 6.25 hours after death and prepared for submicrometer epoxy resin sections and transmission electron microscopy aligned to clinical OCT B-scans. Inside and outside the MNV area, layer thicknesses, phenotypes, and vascular density of native choriocapillaris and neovessels were measured. Lengths of choriocapillaries and intervening gaps in the index eye and in early AMD eyes and healthy eyes with similar age (n= 19 each) from the Project MACULA (Maculopathy Unveiled by Laminar Analysis) online histopathologic resource (http://projectmacula.cis.uab.edu/) were measured with custom software (Caps and Gaps). Descriptive features, vascular density, histologic and OCT layer thicknesses, and distribution of choriocapillaries and intervening gaps. The SIRE correlated to a type 1 MNV that expanded slowly without evidence of exudation and with numerous choroidal vessels traversing Bruch's membrane defects, some visible on OCT. Tissue layers in and adjacent to the MNV area showed continuous RPE and characteristic AMD deposits. Capillary-like neovessels with fenestrations and caveolae resembling native choriocapillaris lined the retinal pigment epithelium (RPE) with a vascular density comparable with surrounding non-MNV areas. Relative to early AMD and healthy aged eyes, the index eye showed similar capillary lengths but larger gaps between vessels, indicating dropout. Outer nuclear layer thickness was preserved and showed less photoreceptor degeneration over areas of relative choriocapillaris health, including the type 1 MNV. Eyes with nonexudative type 1 MNV in AMD may progress to exudation, yet this stable MNV complex supported outer retinal structure for 9 years. Distinguishing features were numerous connecting vessels, high density of neovessels, continuous RPE, and slow growth. Maintaining beneficial type 1 MNV may be a therapeutic strategy.

Quantitative STEM imaging of electron beam induced mass loss of epoxy resin sections

In sample preparation of biological samples for electron microscopy, many types of embedding media are widely used. Unfortunately, none of them is perfectly resistant to beam induced damage. The article is focused on mass loss measuring of pure epoxy resin EMbed 812 that replaced Epon – the most widely used embedding resin for biological electron microscopy, in a form of ultrathin sections with thicknesses ranging from 30 to 100 nm. The STEM imaging was performed in a quantitative way which allowed us to estimate the mass loss directly up to the total dose of 3000 e−/nm2. For data acquisition we used SEM equipped with a commercial STEM detector working at a relatively low acceleration voltage of 30 kV. In this study we estimated the influence of various factors which can affect the endurance of the epoxy resin EMbed 812 ultrathin sections under an electron beam, such as the sample aging, differences between storing the samples in forms of ultrathin sections and whole blocks, ultrathin sections thicknesses, temperature of the sample, probe current, and one or two-sided carbon coating of ultrathin sections. The aim of this work is to investigate beam induced mass loss at electron energies of SEM and find out how to reduce the mass loss.

Morphology of the initial nephron-collecting duct connection in mice using computerized 3D tracing and electron microscopy

Recently, the cellular origin of the connecting tubule (CNT) has been genetically characterized. The CNT is a segment between two embryonically different structures, the collecting duct originating from ureteric bud (UB), and the nephron derived from the cap mesenchyme. However, the cellular detail at the initial connection is limited.The present study demonstrated that the initial connection was composed of cells which were closely associated with the renal vesicle (RV), the initial nephron, and connected with the basal epithelium of the terminal UB tip at discrete points. The identification of the RV and UB tip was based on tracing of tubules on serial epoxy sections at mouse embryonic day 17.5. The cells at the initial connection were characterized by 1) irregularly-shaped nuclei and cells with cytoplasmic processes, 2) electron dense nuclei, 3) abundant intercellular spaces, 4) extensive cell-cell contacts with cell junctions, often zonulae adherences and occasionally focal fusion of opposing plasma membranes, and 5) numerous mitochondria, densely packed rosette-like polyribosomes, and widespread rER in the cytoplasm. Moreover, the tracing revealed that a terminal UB tip frequently connected to two nephrons at different developing stages. The UB tips, the initial connections, and the distal tubules of the S-shaped bodies did not express Na+-Cl− cotransporter, H+-ATPase, or aquaporin 2, while they were expressed in immature CNT of the capillary-loop stage nephrons throughout the kidney development.Consequently, the cells at the initial connection exhibit the morphological features suggestive of energy demanding, protein producing, and intercellular communicating. The cell morphology together with transporter development indicates that these cells serve several functions during the development of the initial connection, and that these functions are different from the cells' final functions as transportation.

Combining high-pressure freezing with pre-embedding immunogold electron microscopy and tomography.

Immunogold labeling of permeabilized whole-mount cells or thin-sectioned material is widely used for the subcellular localization of biomolecules at the high spatial resolution of electron microscopy (EM). Those approaches are well compatible with either 3-dimensional (3D) reconstruction of organelle morphology and antigen distribution or with rapid cryofixation-but not easily with both at once. We describe here a specimen preparation and labeling protocol for animal cell cultures, which represents a novel blend of specifically adapted versions of established techniques. It combines the virtues of reliably preserved organelle ultrastructure, as trapped by rapid freezing within milliseconds followed by freeze-substitution and specimen rehydration, with the advantages of robust labeling of intracellular constituents in 3D through means of pre-embedding NANOGOLD-silver immunocytochemistry. So obtained thin and semi-thick epoxy resin sections are suitable for transmission EM imaging, as well as tomographic reconstruction and modeling of labeling patterns in the 3D cellular context.

Choroidal and Sub-Retinal Pigment Epithelium Caverns: Multimodal Imaging and Correspondence with Friedman Lipid Globules

To survey Friedman lipid globules by high-resolution histologic examination and to compare with multimodal imaging of hyporeflective caverns in eyes with geographic atrophy (GA) secondary to age-related macular (AMD) and other retinal diseases. Histologic survey of donor eyes with and without AMD. Clinical case series with multimodal imaging analysis. Donor eyes (n= 139; 26 with early AMD, 13 with GA, 40 with nAMD, 52 with a healthy macula, and 8 with other or unknown characteristics) and 41 eyes of 28 participants with GA (n= 16), nAMD (n= 8), Stargardt disease (n= 4), cone dystrophy (n= 2), pachychoroid spectrum (n= 6), choroidal hemangioma (n= 1), and healthy eyes (n= 4). Donor eyes were prepared for macula-wide epoxy resin sections through the foveal and perifoveal area. In patients, caverns were identified as nonreflective spaces on OCT images. Multimodal imaging included color and red-free fundus photography; fundus autofluorescence; fluorescein and, indocyanine green angiography; OCT angiography; near-infrared reflectance; and confocal multispectral (MultiColor [Spectralis, Heidelberg Engineering, Germany]) imaging. Presence and morphologic features of globules, and presence and appearance of caverns on multimodal imaging. Globules were found primarily in the inner choroidal stroma (91.0%), but also localized to the sclera (4.9%) and neovascular membranes (2.1%). Mean diameters of solitary and multilobular globules were 58.9±37.8 μm and 65.4±27.9 μm, respectively. Globules showed morphologic signs of dynamism including pitting, dispersion, disintegration, and crystal formation. Evidence for inflammation in the surrounding tissue was absent. En face OCT rendered sharply delimited hyporeflective areas as large as choroidal vessels, frequently grouped around choroid vessels or in the neovascular tissue. Cross-sectional OCT revealed a characteristic posterior hypertransmission. OCT angiography showed absence of flow signal within caverns. Based on prior literature documenting OCT signatures of tissue lipid in atheroma and nAMD, we speculate that caverns are lipid rich. Globules, with similar sizes and tissue locations in AMD and healthy persons, are candidates for histologic correlates of caverns. The role of globules in chorioretinal physiologic features, perhaps as a lipid depot for photoreceptor metabolism, is approachable through clinical imaging.