We report our efforts in identifying optimal scanning laser microscope parameters to study cells in three-dimensional culture. For this purpose we studied contrast of extracellular matrix (ECM) mimics, as well as signal attenuation, and bleaching of red and green fluorescent protein labeled cells. Confocal backscattering, second harmonic generation (SHG), and autofluorescence were sources of contrast in ECM mimics. All common ECM mimics exhibit contrast observable with confocal reflectance microscopy. SHG imaging on collagen I based hydrogels provides high contrast and good optical penetration depth. Agarose is a useful embedding medium because it allows for large optical penetration and exhibits minimal autofluorescence. We labeled breast cancer cells' outline with DsRed2 and nucleus with enhanced green fluorescent protein (eGFP). We observed significant difference both for the bleaching rates of eGFP and DsRed2 where bleaching is strongest during two-photon excitation (TPE) and smallest during confocal imaging. But for eGFP the bleaching rate difference is smaller than for DsRed2. After a few hundred microns depth in a collagen I hydrogel, TPE fluorescence of DsRed2 becomes twice as strong compared to confocal imaging. In fibrin and agarose gels, the imaging depth will need to be beyond 1 mm to notice a TPE advantage.

Vasculogenic erectile dysfunction (ED) is an age-related disease partially dependent on vascular growth factors availability and their specific endothelium receptors activation. Among these, angiopoietins-tyrosine kinase with immunoglobulin and EGF homology domain-2 (Tie2) system seems to be particularly dependent on aging, both in rat and in human corpus cavernosum (CC). Angiopoietins (Ang)1 and 2 are produced by pericytes and endothelial cells respectively, and bind with the same affinity to Tie2 receptor. Ang1 was shown to act as an obligatory agonist inducing Tie2 phosphorylation, which promotes cell-cell and cell-extracellular matrix interactions. On the other hand, Ang2 acts in a context-dependent fashion in the increment or, otherwise, destabilization of pre-existent vasculature.

Interestingly, it has been demonstrated that activation of Tie2 receptor could also be modulated by Tie1, an additional member of the Tie family of receptor tyrosine kinases, and that co-expression of Tie2 is required for Tie1 activation. Indeed, Tie1 similarly to Tie2 is involved in the maintenance of the vascular integrity. Nevertheless, its role in Angiopoietins-Tie system regulation remains poorly understood, and the identification of a true ligand has remained elusive. In the present study, we aim to demonstrate the expression of Tie1 in the endothelium of aged CC in the Rat and Human species.

Twenty-five male Wistar rats were divided in five groups (n=5) and sacrificed by decapitation when they reached the ages of study (6, 12, 18, 24 and 36 months). The penises were excised and immediately fixed in formalin solution. Human CC fragments were obtained from organ donors without known risk factors for ED and divided in two groups: young (16–35 years) and aged (59–74 years). Paraffin embedded sections were deparaffinized, rehydrated and submitted to epitope retrieval with heated Tris-EDTA pH 9.0 buffer before dual-immunolabeling of Tie1 (rabbit anti-Tie1 antibody -abcam) and specific markers of endothelium and smooth muscle cell (goat- anti-PECAM-1 and mouse anti-m-actin antibodies, Santa Cruz Biotechnology Inc. and Millipore, respectively). Appropriate secondary antibodies were employed (anti-rabbit labelled with Alexa 488 with anti-mouse, or, anti –goat labelled with Alexa 568). Nuclei counterstaining was achieved with 4´-6-diamino-2-phenylindole. Images were observed in an Apotome microscope (Carl Zeiss System, Göttingen, Germany) using the filter sets, 01 to DAPI, 09 to Alexa 488 and 31 to Alexa 568, and acquired with Axio Vision 3.0 program (Carl Zeiss System).

Tie1 was detected in the endothelium in the CC of Rat, co-localizing with PECAM-1 (Figure 1a) but not with w-actin (Figure 1b). The intensity of Tie1 immunolabeling was higher in the older animals (24 and 36 months). Tie1 was also detected in Human CC endothelium, suggesting an up-regulation in samples obtained from older individuals (Figure 2a and 2b).

The present results demonstrate for the first time the expression of Tie1 in the endothelium of CC in Rat and Human, which appears to exhibit an age-dependent up-regulation. Ongoing research employing molecular studies will clarify this aspect.

Extended abstract of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.

The ordered L12-type Al3Ti–(8, 10, 15)% Cr intermetallic compounds, namely, Al67Ti25Cr8, Al66Ti24Cr10, and Al59Ti26Cr15, were prepared by induction melting followed by thermomechanical treatment. Their microstructure, compositional variation, and crystal structure were characterized using X-ray diffraction, optical microscopy, and scanning and transmission electron microscopy equipped with energy-dispersive spectroscopy. The Al67Ti25Cr8 alloy consisted of the L12-Al3Ti matrix and precipitates of α2-Ti3Al, D022-Al3Ti, and γ-TiAl. The Al66Ti24Cr10 and Al59Ti26Cr15 alloys consisted of the L12-Al3Ti matrix and grains of α-TiAl and β-Cr.

X-ray fluorescence (XRF) microscopy is an important tool for studying trace metals in biology, enabling simultaneous detection of multiple elements of interest and allowing quantification of metals in organelles without the need for subcellular fractionation. Currently, analysis of XRF images is often done using manually defined regions of interest (ROIs). However, since advances in synchrotron instrumentation have enabled the collection of very large data sets encompassing hundreds of cells, manual approaches are becoming increasingly impractical. We describe here the use of soft clustering to identify cell ROIs based on elemental contents, using data collected over a sample of the malaria parasite Plasmodium falciparum as a test case. Soft clustering was able to successfully classify regions in infected erythrocytes as “parasite,” “food vacuole,” “host,” or “background.” In contrast, hard clustering using the k-means algorithm was found to have difficulty in distinguishing cells from background. While initial tests showed convergence on two or three distinct solutions in 60% of the cells studied, subsequent modifications to the clustering routine improved results to yield 100% consistency in image segmentation. Data extracted using soft cluster ROIs were found to be as accurate as data extracted using manually defined ROIs, and analysis time was considerably improved.

Author names are referenced by their paper numbers.

Since it was discovered in 2004, graphene has attracted enormous attention as an emerging material for future devices, but it has been found that conventional lithographic processes based on polymer resist degrade its intrinsic performance. Recently, our group studied a resist-free scanning tunneling microscopy-based lithography in various atmospheres by injecting volatile liquids into a chamber. In this study, multilayer graphene was scanned and etched by controlling bias voltage under methanol pressure. We focused on improving patterning results in terms of depth and line width, while the previous study was performed to find an optimum gas environment for patterning on a graphite surface. Specifically, we report patterning outputs depending on conditions of voltage, current, and pressure. The optimum conditions for methanol environment etching were a gas pressure in the range of 41–50 torr, a −4 V tip bias, and a 2 nA tunneling current.

A need exists to prolong the release of rapidly metabolized peptides of a low molecular weight, while delivering this peptide without environmental interference. Previous studies have used bovine serum albumin (BSA) as a model peptide to study release characteristics from alginate microcapsules. BSA is 66 kDa in size, while the peptide of interest here, connexin-43 carboxyl-terminus mimetic peptide (αCT1), is only 3.4 kDa. Such a change in size results in a much different set of release parameters. Our overall goal is a sustained release over a 24+ h period. Prolonged application of the peptide to a wound site to investigate therapeutic effects is ideal. As a result, a diffusion method using alginate microcapsules, along with the addition of poly-l-lysine and poly-l-ornithine, has been explored. We first aimed to establish and characterize our parameters through a set of parametric tests. Variations in polymer coating, change in pH, and changes in loading ratio have previously been shown to effect release using model compounds. Here we test specific changes in these parameters to show effects on the release of αCT1. Additionally, the microcapsules were attached to several biomaterials and surgical implants by ultraviolet cross-linking to study the effectiveness of attachment and delivery. Analysis and measurements using phase contrast microscopy, scanning electron microscopy, and atomic force microscopy were used to characterize changes in microcapsule morphology.

Pole figures are often used to present crystal orientation data. The huge number of single orientation measurements acquired by electron backscatter diffraction (EBSD) poses a challenge for pole figure representation due to the large number of calculations required. This significantly reduces the speed at which the data may be rotated and affects the ability to switch between different projection types. In the present work, it will be shown that satisfactory representation of orientation data in different projection types can generally be achieved by an imaging of a spherical projection. With this approach, explicit calculation of the projections is no longer required, allowing for both real-time dataset rotation and real-time switching between all projection types relevant to materials science. The technique can be applied to any other directional property distribution, for example, not only for EBSD orientation presentation.

Extended abstract of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.

Paleopathology, summarily defined as the study of past diseases, has on the differential diagnosis a major challenge. Histological techniques offered the possibility to look inside the microstructure of both normal and abnormal body tissues to diagnose diseases that affected past populations, leading to the development of a new field of research - paleohistology or paleohistopathology. However, and contrary to paleopathology whose journey is well-established, in paleohistopathology there are many gaps that need to be filled. This occurrence is probably the result of a nonsystematic and non-standardized approach to the microscopic study of skeletal abnormalities, especially those of infectious origin involving periosteal new bone formation (PNBF). The aims of this work were: (1) to search for differences in the microstructure of PNBF with regard to the cause of death of the individual; (2) to infer differences between the macroscopic and microscopic proprieties of bone lesions, and (3) to ascertain the impact of diagenetic changes in the bone microstructure. For histological examination under transmitted and polarized light, a total of 34 dry bone specimens: 26 belonging to 23 individuals from the Human Identified Skeletal Collection from the Bocage Museum (Lisbon, Portugal), and eight from archaeological skeletons were prepared. The documented bone samples were collected from individuals who died from tuberculosis-TB (Group 1), non-TB infectious diseases (Group 2), and conditions other than those of TB and non-TB infectious origin (Group 3).

With regard to the diagnosis of pathological conditions, differences in the microstructure of PNBF were seen between Group 1 and Group 2 of cause of death and within groups. Multiple layers of “appositional bone” enclosing numerous primary vascular canals were the pattern most commonly observed (n=4) in the samples from Group 1. This type of PNBF seems to mimic the appositional growth that characterizes the modeling process of the periosteal and endosteal membranes (PEM) during rapid growth periods. An abnormal stimulation of growth factors, especially of the vascular endothelial growth factor (VEGF) may eventually explain the extensive hypervascularization observed (Fig. 1 A-B). Periosteal lesions on ribs are normally associated with pulmonary infection (e.g. TB) disseminated from the lungs (via pleura) to the ribs. However, three samples (one from Group 1, two from Group 2) presented a microstructure compatible with subperiosteal hematomas (Fig. 1 C-D). Repetitive microtrauma (e.g. chest wall vibration) that causes detachment of the periosteum may have leaded to subperiosteal bleeding and hematoma formation. These observations suggest that beyond pulmonary diseases other mechanisms may stimulate PNBF on the visceral surface of ribs. Histological analysis was also fundamental in the description and characterization of bone changes. For example, of the five samples with “consolidated” fracture callus, only two presented a truly mature and remodeled microstructure. This means that the outer surface of a bone lesion may not give a complete picture of the tissues response (Fig. 2 A-B). In spite of the good preservation of some bone samples, massive diagenetic changes due to the action of bacteria and fungi were observed at microscopic level. This clearly suggests that gross inspection is not a good measure of the bone tissue quality. In contrast, microscopy is essential to differentiate between pseudopathology and physiological or pathological signs.

Microscopy revealed surprising results that reinforce the pertinence of applying histological techniques in the description and diagnosis of bone changes in human remains.

This research was supported by Fundação para a Ciência e Tecnologia, Portugal [SFRH/BD/36739/2007, Sandra Assis].

The use of nanoparticles for the diagnosis and treatment of cancer requires the complete characterization of their toxicity, including accurately locating them within biological tissues. Owing to their size, traditional light microscopy techniques are unable to resolve them. Transmission electron microscopy provides the necessary spatial resolution to image individual nanoparticles in tissue, but is severely limited by the very small analysis volume, usually on the order of tens of cubic microns. In this work, we developed a scanning transmission electron microscopy (STEM) approach to analyze large volumes of tissue for the presence of polyethylene glycol-coated Raman-active-silica-gold-nanoparticles (PEG-R-Si-Au-NPs). This approach utilizes the simultaneous bright and dark field imaging capabilities of STEM along with careful control of the image contrast settings to readily identify PEG-R-Si-Au-NPs in mouse liver tissue without the need for additional time-consuming analytical characterization. We utilized this technique to analyze 243,000 μm3 of mouse liver tissue for the presence of PEG-R-Si-Au-NPs. Nanoparticles injected into the mice intravenously via the tail vein accumulated in the liver, whereas those injected intrarectally did not, indicating that they remain in the colon and do not pass through the colon wall into the systemic circulation.

High-chromium heat-resistant steel has been widely used as the key material to improve the condition of steam pressure and temperature in the modern high-efficiency power plants. Despite the use of the steel above 550°C for several decades, its major failure is owing to the creep fracture. In this study, the effect of creep stress on the microstructure in 9–12% Cr steel has been investigated microscopically, and it is clarified that the creep stress enhances precipitation of Laves phase and influences the lath width and dislocation density in lath interior.

The ability to correctly track objects in time-lapse sequences is important in many applications of microscopy. Individual object motions typically display a level of dynamic regularity reflecting the existence of an underlying physics or biology. Best results are obtained when this local information is exploited. Additionally, if the particle number is known to be approximately constant, a large number of tracking scenarios may be rejected on the basis that they are not compatible with a known maximum particle velocity. This represents information of a global nature, which should ideally be exploited too. Some time ago, we devised an efficient algorithm that exploited both types of information. The tracking task was reduced to a max-flow min-cost problem instance through a novel graph structure that comprised vertices representing objects from three consecutive image frames. The algorithm is explained here for the first time. A user-friendly implementation is provided, and the specific relaxation mechanism responsible for the method's effectiveness is uncovered. The software is particularly competitive for complex dynamics such as dense antiparallel flows, or in situations where object displacements are considerable. As an application, we characterize a remarkable vortex structure formed by bacteria engaged in interstitial motility.

III-V nitride (AlGa)N distributed Bragg reflector devices are characterized by combined high-angle annular dark-field (HAADF) and electron energy loss spectroscopy (EELS) in the scanning transmission electron microscope. Besides the complete structural characterization of the AlN and GaN layers, the formation of AlGaN transient layers is revealed using Vegard law on profiles of the position of the bulk plasmon peak maximum. This result is confirmed by comparison of experimental and simulated HAADF intensities. In addition, we present an advantageous method for the characterization of nano-feature structures using low-loss EELS spectrum image (EEL-SI) analysis. Information from the materials in the sample is extracted from these EEL-SI at high spatial resolution.The log-ratio formula is used to calculate the relative thickness, related to the electron inelastic mean free path. Fitting of the bulk plasmon is performed using a damped plasmon model (DPM) equation. The maximum of this peak is related to the chemical composition variation using the previous Vegard law analysis. In addition, within the context of the DPM, information regarding the structural properties of the material can be obtained from the lifetime of the oscillation. Three anomalous segregation regions are characterized, revealing formation of metallic Al islands.

Membranoproliferative glomerulonephritis (MPGN) encompasses 7 to 10% of all biopsied glomerulonephritis. They are divided in: MPGN type I; MPGN type II and MPGN type III, being primary or secondary. MPGN type I are the most frequent, MPGN types II and III are very rare and difficult to diagnose without clinical and morphologic findings integration. MPGN type II or Dense Deposit Disease has a varied morphologic appearance with a few numbers of cases showing a membranoproliferative pattern by Light microscopy (LM). Electron microscopy (EM) is pivotal to confirm the diagnosis.

We present a case of 35 years old man, with nephrotic proteinuria and mild renal insufficiency since 2 years. The only relevant clinical data is facial lipodystrophy. Complement 3 (C3) was low and C3 nephritic factor negative. There were not other relevant abnormalities. Renal biopsy was fixed in buffered formaldehyde 10% and performed for LM. The frozen fragment, prepared for observation by fluorescence microscopy - immunofluorescence (IMF) -, was prepared to be stained with florescent anti-serums, against immunoglobulines (IgG, IgA and IgM) and complement factors (C3, C4, and C1q). EM was later done on tissue formaldehyde fixed reprocessed from paraffin-embebbed for LM, because there was no tissue fragment fixed in glutaraldehyde.

LM showed variable endocapillary hypercelullarity, with neutrophils infiltration. Capillary walls were thickened due to the deposition of elongate and ribbon-like deposits. Few double contours were visible (Figure 1a). IMF demonstrated the presence of C3 deposits in the capillary walls and mesangium (Figure 1b). EM confirmed the presence of an intramembranous dense deposit along basement membrane which was thickened (Figure 1c). LM and IMF findings favored the diagnosis of MPGN type II with C3 deposits and thickening of basement membrane. Nevertheless EM was essential to confirm intramembranous unequivocally dense deposits.

MPGN type II is a rare glomerulonephritis mediated by complement deregulation. The integration of clinical and morphologic findings is essential to get a correct diagnosis. In this setting EM is highly distinctive and required for a definitive diagnosis.

This study investigated the inflammatory effect of intraoperative mitomycin C (MMC) on adhesion reformation in human rectus muscles. Ten consecutive patients who underwent medial rectus resection had their postoperative rectus muscles divided into two groups: control group (n = 10) and MMC group (n = 10). In the MMC group, the muscle was soaked for 2 min with MMC, prepared as a 0.2 mg/mL (0.02%) solution. The 0.02% MMC reactions were examined using histological analysis with hematoxylin-eosin (inflammatory response) and Masson's trichrome (collagen fibrils), immunoreactivities of cyclooxygenase-II (inflammatory response), and collagen type I and III, scanning electron microscopy analysis to quantify the diameter and D-periodicity of collagen fibrils, and atomic force microscopy analysis to quantify the diameter, D-periodicity, and adhesion force of collagen fibrils. The rectus muscles treated with 0.02% MMC showed a significantly increased inflammatory response (p < 0.05), increased collagen density (p < 0.0001), increased fibril diameter (p < 0.001 or p < 0.05), and decreased fibril adhesion force (p < 0.005) compared to the rectus muscles in the control group. MMC simultaneously caused an inflammatory response as well as nanostructural and biomechanical property changes in the collagen fibril network.

Uni- and biaxial tension deformation tests, with different degrees of deformation, have been done on AISI 430 (EN 1.4016) ferritic stainless steel samples, which had both different chemical compositions and had undergone different annealing treatments. The initial and deformed materials were characterized by using electron backscatter diffraction and backscatter electron imaging in a scanning electron microscope together with electron probe microanalysis. The correlation observed among the chemical compositions, annealing treatment, and strain level obtained after deformation is discussed.