Comparison of 3D cellular imaging techniques based on scanned electron probes: Serial block face SEM vs. Axial bright-field STEM tomography.

Microscopies based on focused electron probes allow the cell biologist to image the 3D ultrastructure of eukaryotic cells and tissues extending over large volumes, thus providing new insight into the relationship between cellular architecture and function of organelles. Here we compare two such techniques: electron tomography in conjunction with axial bright-field scanning transmission electron microscopy (BF-STEM), and serial block face scanning electron microscopy (SBF-SEM). The advantages and limitations of each technique are illustrated by their application to determining the 3D ultrastructure of human blood platelets, by considering specimen geometry, specimen preparation, beam damage and image processing methods. Many features of the complex membranes composing the platelet organelles can be determined from both approaches, although STEM tomography offers a higher ∼3 nm isotropic pixel size, compared with ∼5 nm for SBF-SEM in the plane of the block face and ∼30 nm in the perpendicular direction. In this regard, we demonstrate that STEM tomography is advantageous for visualizing the platelet canalicular system, which consists of an interconnected network of narrow (∼50-100 nm) membranous cisternae. In contrast, SBF-SEM enables visualization of complete platelets, each of which extends ∼2 µm in minimum dimension, whereas BF-STEM tomography can typically only visualize approximately half of the platelet volume due to a rapid non-linear loss of signal in specimens of thickness greater than ∼1.5 µm. We also show that the limitations of each approach can be ameliorated by combining 3D and 2D measurements using a stereological approach.

Development, income transfer strategies, and the nutritional transition in Brazilian children from a rural and remote region.

INTRODUCTION: Global development processes have been associated with the nutritional transition, where undernutrition is replaced by overnutrition. Income transfer policies in Brazil have targeted hunger, but may not address the need for balanced nutrition. METHODS: Data was collected from government databanks that document the nutritional status of Brazilians applying for social services. This data was analyzed for descriptive statistics. RESULTS: Development and income transfer processes appear to be associated with an increase in overweight children between the years 2008 and 2012. CONCLUSIONS: Income transfer programs need to incorporate educational programs that address the need to budget for balanced nutrition.

High-resolution structural profile of hylaseptin-4: Aggregation, membrane topology and pH dependence of overall membrane binding process.

Hylaseptin-4 (HSP-4, GIGDILKNLAKAAGKAALHAVGESL-NH2) is an antimicrobial peptide originally isolated from Hypsiboas punctatus tree frog. The peptide has been chemically synthetized for structural investigations by CD and NMR spectroscopies. CD experiments reveal the high helical content of HSP-4 in biomimetic media. Interestingly, the aggregation process seems to occur at high peptide concentrations either in aqueous solution or in presence of biomimetic membranes, indicating an increase in the propensity of the peptide for adopting a helical conformation. High-resolution NMR structures determined in presence of DPC-d38 micelles show a highly ordered α-helix from amino acid residues I2 to S24 and a smooth bend near G14. A large separation between hydrophobic and hydrophilic residues occurs up to the A16 residue, from which a shift in the amphipathicity is noticed. Oriented solid-state NMR spectroscopy show a roughly parallel orientation of the helical structure along the POPC lipid bilayer surface, with an insertion of the hydrophobic N-terminus into the bilayer core. Moreover, a noticeable pH dependence of the aggregation process in both aqueous and in biomimetic membrane environments is attributed to a single histidine residue (H19). The protonation degree of the imidazole side-chain might help in modulating the peptide-peptide or peptide-lipid interactions. Finally, molecular dynamics simulations confirm the orientation and preferential helical conformation and in addition, show that HSP-4 tends to self-aggregate in order to stabilize its active conformation in aqueous or phospholipid bilayer environments.

Limitations of beam damage in electron spectroscopic tomography of embedded cells.

Elemental mapping in the energy filtering transmission electron microscope (EFTEM) can be extended into three dimensions (3D) by acquiring a series of two-dimensional (2D) core-edge images from a specimen oriented over a range of tilt angles, and then reconstructing the volume using tomographic methods. EFTEM has been applied to imaging the distribution of biological molecules in 2D, e.g. nucleic acid and protein, in sections of plastic-embedded cells, but no systematic study has been undertaken to assess the extent to which beam damage limits the available information in 3D. To address this question, 2D elemental maps of phosphorus and nitrogen were acquired from unstained sections of plastic-embedded isolated mouse thymocytes. The variation in elemental composition, residual specimen mass and changes in the specimen morphology were measured as a function of electron dose. Whereas 40% of the total specimen mass was lost at doses above 10(6) e(-)/nm(2), no significant loss of phosphorus or nitrogen was observed for doses as high as 10(8) e(-)/nm(2). The oxygen content decreased from 25 + or - 2 to 9 + or - 2 atomic percent at an electron dose of 10(4) e(-)/nm(2), which accounted for a major component of the total mass loss. The specimen thickness decreased by 50% after a dose of 10(8) e(-)/nm(2), and a lateral shrinkage of 9.5 + or - 2.0% occurred from 2 x 10(4) to 10(8) e(-)/nm(2). At doses above 10(7) e(-)/nm(2), damage could be observed in the bright field as well in the core edge images, which is attributed to further loss of oxygen and carbon atoms. Despite these artefacts, electron tomograms obtained from high-pressure frozen and freeze-substituted sections of C. elegans showed that it is feasible to obtain useful 3D phosphorus and nitrogen maps, and thus to reveal quantitative information about the subcellular distributions of nucleic acids and proteins.

Monte Carlo electron-trajectory simulations in bright-field and dark-field STEM: implications for tomography of thick biological sections.

A Monte Carlo electron-trajectory calculation has been implemented to assess the optimal detector configuration for scanning transmission electron microscopy (STEM) tomography of thick biological sections. By modeling specimens containing 2 and 3 at% osmium in a carbon matrix, it was found that for 1-microm-thick samples the bright-field (BF) and annular dark-field (ADF) signals give similar contrast and signal-to-noise ratio provided the ADF inner angle and BF outer angle are chosen optimally. Spatial resolution in STEM imaging of thick sections is compromised by multiple elastic scattering which results in a spread of scattering angles and thus a spread in lateral distances of the electrons leaving the bottom surface. However, the simulations reveal that a large fraction of these multiply scattered electrons are excluded from the BF detector, which results in higher spatial resolution in BF than in high-angle ADF images for objects situated towards the bottom of the sample. The calculations imply that STEM electron tomography of thick sections should be performed using a BF rather than an ADF detector. This advantage was verified by recording simultaneous BF and high-angle ADF STEM tomographic tilt series from a stained 600-nm-thick section of C. elegans. It was found that loss of spatial resolution occurred markedly at the bottom surface of the specimen in the ADF STEM but significantly less in the BF STEM tomographic reconstruction. Our results indicate that it might be feasible to use BF STEM tomography to determine the 3D structure of whole eukaryotic microorganisms prepared by freeze-substitution, embedding, and sectioning.

Determination of quantitative distributions of heavy-metal stain in biological specimens by annular dark-field STEM.

It is shown that dark-field images collected in the scanning transmission electron microscope (STEM) at two different camera lengths yield quantitative distributions of both the heavy and light atoms in a stained biological specimen. Quantitative analysis of the paired STEM images requires knowledge of the elastic scattering cross sections, which are calculated from the NIST elastic scattering cross section database. The results reveal quantitative information about the distribution of fixative and stain within the biological matrix, and provide a basis for assessing detection limits for heavy-metal clusters used to label intracellular proteins. In sectioned cells that have been stained only with osmium tetroxide, we find an average of 1.2+/-0.1 Os atom per nm(3), corresponding to an atomic ratio of Os:C atoms of approximately 0.02, which indicates that small heavy atom clusters of Undecagold and Nanogold can be detected in lightly stained specimens.

Reprint of "Three-dimensional elemental mapping of phosphorus by quantitative electron spectroscopic tomography (QuEST)" [J. Struct. Biol. 160 (2007) 35-48].

We describe the development of quantitative electron spectroscopic tomography (QuEST), which provides 3-D distributions of elements on a nanometer scale. Specifically, it is shown that QuEST can be applied to map the distribution of phosphorus in unstained sections of embedded cells. A series of 2-D elemental maps is derived from images recorded in the energy filtering transmission electron microscope for a range of specimen tilt angles. A quantitative 3-D elemental distribution is then reconstructed from the elemental tilt series. To obtain accurate quantitative elemental distributions it is necessary to correct for plural inelastic scattering at the phosphorus L(2,3) edge, which is achieved by acquiring unfiltered and zero-loss images at each tilt angle. The data are acquired automatically using a cross correlation technique to correct for specimen drift and focus change between successive tilt angles. An algorithm based on the simultaneous iterative reconstruction technique (SIRT) is implemented to obtain quantitative information about the number of phosphorus atoms associated with each voxel in the reconstructed volume. We assess the accuracy of QuEST by determining the phosphorus content of ribosomes in a eukaryotic cell, and then apply it to estimate the density of nucleic acid in chromatin of the cell's nucleus. From our experimental data, we estimate that the sensitivity for detecting phosphorus is 20 atoms in a 2.7 nm-sized voxel.

Reprint of "On the feasibility of visualizing ultrasmall gold labels in biological specimens by STEM tomography" [J. Struct. Biol. 159 (2007) 507-522].

Labeling with heavy atom clusters attached to antibody fragments is an attractive technique for determining the 3D distribution of specific proteins in cells using electron tomography. However, the small size of the labels makes them very difficult to detect by conventional bright-field electron tomography. Here, we evaluate quantitative scanning transmission electron microscopy (STEM) at a beam voltage of 300kV for detecting 11-gold atom clusters (Undecagold) and 1.4nm-diameter nanoparticles (Nanogold) for a variety of specimens and imaging conditions. STEM images as well as tomographic tilt series are simulated by means of the NIST Elastic-Scattering Cross-Section Database for gold clusters embedded in carbon. The simulations indicate that the visibility in 2D of Undecagold clusters in a homogeneous matrix is maximized for low inner collection semi-angles of the STEM annular dark-field detector (15-20mrad). Furthermore, our calculations show that the visibility of Undecagold in 3D reconstructions is significantly higher than in 2D images for an inhomogeneous matrix corresponding to fluctuations in local density. The measurements demonstrate that it is possible to detect Nanogold particles in plastic sections of tissue freeze-substituted in the presence of osmium. STEM tomography has the potential to localize specific proteins in permeabilized cells using antibody fragments tagged with small heavy atom clusters. Our quantitative analysis provides a framework for determining the detection limits and optimal experimental conditions for localizing these small clusters.

Microglia and neurons in the hippocampus of migratory sandpipers.

The semipalmated sandpiper Calidris pusilla and the spotted sandpiper Actitis macularia are long- and short-distance migrants, respectively. C. pusilla breeds in the sub-arctic and mid-arctic tundra of Canada and Alaska and winters on the north and east coasts of South America. A. macularia breeds in a broad distribution across most of North America from the treeline to the southern United States. It winters in the southern United States, and Central and South America. The autumn migration route of C. pusilla includes a non-stop flight over the Atlantic Ocean, whereas autumn route of A. macularia is largely over land. Because of this difference in their migratory paths and the visuo-spatial recognition tasks involved, we hypothesized that hippocampal volume and neuronal and glial numbers would differ between these two species. A. macularia did not differ from C. pusilla in the total number of hippocampal neurons, but the species had a larger hippocampal formation and more hippocampal microglia. It remains to be investigated whether these differences indicate interspecies differences or neural specializations associated with different strategies of orientation and navigation.

Bilateral arteriovenous malformations: case report.

The authors describe a case of supratentorial bilateral arteriovenous malformations in a patient who suffered an intracerebral hemorrhage. It was radiologically demonstrated that the arteriovenous malformations were separate and bilateral, located in the temporal lobes. They were removed in two operations with preoperative embolization of one. The patient did well with no neurological damage.

Effect of sodium carboxymethylcellulose and methylprednisolone on the healing of jejunal anastomoses in rats.

Sodium carboxymethylcellulose (SCMC) has been effective in reducing adhesion formation and corticosteroids reduce the inflammatory process. The objective of this study was to define the intraperitoneal (ip) effects of SCMC combined with intramuscular (im) methylprednisolone on peritoneal adhesion formation and on jejunal anastomosis healing in rats. Twenty Wistar rats (200-350 g) were divided into four groups (N = 5): groups I and III (controls) 5 and 21 days of treatment before sacrifice, respectively; groups II and IV (experimental groups) 5 and 21 days of treatment, respectively. SCMC (1%) was infused into the abdominal cavity and methylprednisolone (10 mg kg-1 day-1) was injected im daily from the day before surgery for animals of groups II and IV. All rats were submitted to a jejunal anastomosis. Sections of the anastomosis were prepared for routine histopathological analysis. The abdominal adhesion of group IV was less intense when compared with group III (P<0.0008). Anastomotic resistance was higher in groups II and IV when compared with groups I and III, respectively (P<0.05). There was no histological difference between groups I and II (exuberant granulation tissue on the serosal surface). Group III presented little peritoneal fibrinous tissue, with numerous thick collagen fibers. Group IV presented extensive although immature young fibrous tissue with rare thick collagen fibers. Sodium carboxymethylcellulose combined with corticosteroids seemed to diminish peritoneal adhesion but did not reduce anastomotic resistance.

Influence of acute renal failure on the mononuclear phagocytic system.

Several studies show the ability of macrophages to remove particles injected into the bloodstream. This function seems to be increased in the presence of acute renal failure. The objective of the present study was to assess the phagocytic function of the main organs (spleen, liver and lung) of the mononuclear phagocytic system in renal and postrenal failures. Fifteen rats (250-350 g) were divided into three groups (N = 5): group I - control; group II - ligature of both ureters, and group III - bilateral nephrectomy. On the third postoperative day, all animals received an iv injection of 1 ml/kg 99mTc sulfur colloid. Blood samples were collected for the assessment of plasma urea, creatinine, sodium, and potassium concentrations and arterial gasometry. Samples of liver, spleen, lung and blood clots were obtained and radioactivity was measured. Samples of liver, spleen, lung and kidney were prepared for routine histopathological analysis. Plasma urea, creatinine and potassium concentrations in groups II and III were higher than in group I (P<0.05). Plasma sodium concentrations in groups II and III were lower than in group I (P<0.05). Compensated metabolic acidosis was observed in the presence of postrenal failure. Group II animals showed a lower level of radioactivity in the spleen (0.98) and lung (2.63), and a higher level in the liver (105.51) than control. Group III animals showed a lower level of radioactivity in the spleen (11.94) and a higher level in the liver (61.80), lung (11.30) and blood clot (5.13) than control. In groups II and III liver steatosis and bronchopneumonia were observed. Renal and postrenal failures seem to interfere with blood clearance by the mononuclear phagocytic system.

Distal anterior cerebral artery aneurysms.

BACKGROUND: The incidence of saccular aneurysms in the distal anterior cerebral artery (DACA aneurysms), also called pericallosal or A2 aneurysms, has been estimated to be from 1.5 to 9.0% of all intracranial aneurysms in large series in the literature [5,10,12,18]. All reported series of DACA aneurysms have shown a high association with intracranial aneurysms in other locations. These aneurysms are fragile, frequently rupture prematurely during exposure, and have a higher morbidity than expected from their angiographic appearance and location. METHODS: A total of 1,350 patients with cerebral aneurysms were operated in the Department of Neurosurgery at Santa Casa Hospital in Belo Horizonte from January 1982 to January 1998. Seventy-two of those 1,350 patients had DACA aneurysms (5.3%), 51 female and 21 male. The age ranged from 26 to 69 years, the mean age being 44 years. This group of patients is reported herein. We propose three different approaches to DACA aneurysms depending on their location, all of them performed through a unilateral triangular bone flap that can then vary from fronto-basal to parietal. RESULTS: There was no mortality among patients with a single DACA aneurysm. Five deaths in this series were cases of multiple aneurysms. In patients with preoperative Hunt and Kosnick grades I and II [9], we had 90% good results. In grade III patients, we achieved 68.4% good results. The only grade IV patient had a full recovery. All 74 DACA aneurysms in the 72 operated patients were completely clipped, including two giant aneurysms. CONCLUSION: DACA aneurysms have higher morbidity and mortality rates when compared to other supratentorial aneurysms. We recommend the use of a unilateral interhemispheric approach as the most appropriate for aneurysms in this location. In cases of patients with multiple aneurysms who require two different craniotomies, we propose two surgical stages, starting with the aneurysm that has bled. A high mortality rate was noted when two craniotomies were performed in the same sitting.

Giant posterior cerebral artery aneurysm in a 4-year-old child: case report.

Posterior cerebral artery aneurysms in children aged 5 years or less are very rare. We were able to find only 10 cases previously described in the literature. We present a case of giant bilobulated aneurysm of the distal right posterior cerebral artery in a 4-year-old child with an initial history of spontaneous subarachnoid hemorrhage. The aneurysm was clipped and the patient had full recovery. Based on the literature review, we discuss the characteristics of these rare aneurysms, making a comparison with different age groups.

Congenital inclusion cysts of the anterior fontanelle.

BACKGROUND: Congenital inclusion cysts of the anterior fontanelle are rare lesions. Both dermoid and epidermoid cysts are located in the midline of the scalp and occupy the subgaleal space. METHODS: We report 7 cases, 4 boys (57.1%), and 3 girls (42.9%), with ages ranging from 3 months to 16 years (mean 40.85 +/- 68.56 months; median 10 months). Four patients (57.1%) were white and 3 (42.9%) were Afro-Brazilian. RESULTS: The cysts had manifested soon after birth in all patients. They gradually enlarged, with no intracranial extensions. Four patients had dermoid cysts and the others had epidermoid cysts. All of them underwent surgery with complete excision of the cyst and no capsular rupture. There were no complications and no deaths. CONCLUSIONS: Congenital inclusion cysts of the anterior fontanelle are rare lesions that usually manifest at birth. The diagnosis is usually easy and surgery is mandatory, with a good prognosis. Recurrence is rare.

Chagas seropositive donors in kidney transplantation.

Among 239 kidney transplantations between September 1992 and September 1997, nine recipients' kidneys were obtained from Chagas seropositive donors. All of these patients were treated with benznidazole (5 mg/kg/d) for 14 days starting on day 0. None of them experienced acute Chagas disease or seroconversion even after 10 years follow-up. We concluded that, due to cadaver shortage, donors with positive serology for Chagas disease should not be excluded.

Basilar bifurcation aneurysms.

Direct operative management of basilar bifurcation aneurysms is one of the most challenging procedures in the realm of vascular neurosurgery, as these lesions are deeply situated in the interpeduncular region and maintain an intimate relationship with important anatomical structures. Direct clipping of aneurysms generally represents the gold standard of surgical treatment, as it allows exclusion of the aneurysmal sac from the circulation, evacuation of aneurysmal contents for decompression, and preservation of efferent flow. The author describes his experience with 123 operated patients of basilar bifurcation aneurysms from January 1977 till December 1995. In these 123 consecutive surgeries the results were 85% good outcome, 8.1% morbidity, and 6.5% mortality. In the first years of this series the pterional or subtemporal approaches were used, depending on the level of the basilar bifurcation, the exact origin of the sac, its projection and size. From 1987 on all patients were operated by a modified pterional approach described initially by Sano as temporopolar approach.

Carotid endarterectomy under regional anesthesia.

Regional anesthesia for carotid endarterectomy is a simple, reliable, and virtually complication-free technique. We began to perform a series of carotid endarterectomy under regional anesthesia at our institution in May 1990. This report describes our experience with 180 operated patients from May 1990 till December 1995, with regional anesthesia. All patients were operated with microsurgery and we utilized the deeply cervical plexus block at the C-4 level associated with superficial block, along the posterior border of the externocleidomastoid muscle. The main advantage of this technique of anesthesia is that it is the only exact method of assessing the need of a shunt by using the neurological status of the awaken patient during trial carotid cross-clamping. The regional anesthesia allows carotid endarterectomy to be safely performed on patients with advanced cardiac disease or severe chronic obstructive pulmonary disease who were not good candidates for general anesthesia. In this 180 patients we performed 198 consecutive endarterectomies (10% bilateral) with a total morbidity-mortality rate of 2.0%.

[Ganglioglioma of the pineal region: case report]. / Ganglioglioma da região da pineal: relato de caso.

Ganglioglioma are tumors presenting neoplastic glial cells and nerve cells, very rarely found in the pineal region. Only eight cases have been previously published in the literature. We present the case of a 14 years-old male patient with a ganglioglioma of the pineal region. The patient was treated surgically by a suboccipital transtentorial approach with complete removal. Histopathologic specimens with immunostaining revealed a ganglioglioma grade I. We conclude that these tumors are rare and should be treated surgically aiming total remove. If it is not possible or in case of reocurrence the patient should be followed and radiotherapy could be considered.

[Lipoma of the cerebellopontine angle: case report]. / Lipoma do ângulo pontocerebelar: relato de caso.

Lipoma of the cerebellopontine angle is a very rare tumor. We report the case of a 14-years-old female, with left side deafness during three years, associated with headache. CT scan showed an hypodense mass, without enhancement at the cerebellopontine angle. The patient was treated surgically by left retrosigmoid approach. The lesion involved the eighth and seventh cranial nerves and only a partial removal was performed. The postoperative course was uneventful. She had no more headache; the deafness of the left side remained unchanged. Asymptomatic lipoma of the cerebellopontine angle can be treated conservatively, although those with progressive symptoms should be treated surgically, with total or partial remove based on their neurovascular involvement.