Vesicular trafficking of immune mediators in human eosinophils revealed by immunoelectron microscopy

Energy Technology Data Exchange (ETDEWEB)

Melo, Rossana C.N., E-mail: rossana.melo@ufjf.edu.br [Laboratory of Cellular Biology, Department of Biology, ICB, Federal University of Juiz de Fora, UFJF, Rua José Lourenço Kelmer, Juiz de Fora, MG 36036-900 (Brazil); Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, CLS 943, Boston, MA 02215 (United States); Weller, Peter F. [Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, CLS 943, Boston, MA 02215 (United States)

2016-10-01

Electron microscopy (EM)-based techniques are mostly responsible for our current view of cell morphology at the subcellular level and continue to play an essential role in biological research. In cells from the immune system, such as eosinophils, EM has helped to understand how cells package and release mediators involved in immune responses. Ultrastructural investigations of human eosinophils enabled visualization of secretory processes in detail and identification of a robust, vesicular trafficking essential for the secretion of immune mediators via a non-classical secretory pathway associated with secretory (specific) granules. This vesicular system is mainly organized as large tubular-vesicular carriers (Eosinophil Sombrero Vesicles – EoSVs) actively formed in response to cell activation and provides a sophisticated structural mechanism for delivery of granule-stored mediators. In this review, we highlight the application of EM techniques to recognize pools of immune mediators at vesicular compartments and to understand the complex secretory pathway within human eosinophils involved in inflammatory and allergic responses. - Highlights: • Application of EM to understand the complex secretory pathway in human eosinophils. • EM techniques reveal an active vesicular system associated with secretory granules. • Tubular vesicles are involved in the transport of granule-derived immune mediators.

Vesicular trafficking of immune mediators in human eosinophils revealed by immunoelectron microscopy

International Nuclear Information System (INIS)

Melo, Rossana C.N.; Weller, Peter F.

2016-01-01

Electron microscopy (EM)-based techniques are mostly responsible for our current view of cell morphology at the subcellular level and continue to play an essential role in biological research. In cells from the immune system, such as eosinophils, EM has helped to understand how cells package and release mediators involved in immune responses. Ultrastructural investigations of human eosinophils enabled visualization of secretory processes in detail and identification of a robust, vesicular trafficking essential for the secretion of immune mediators via a non-classical secretory pathway associated with secretory (specific) granules. This vesicular system is mainly organized as large tubular-vesicular carriers (Eosinophil Sombrero Vesicles – EoSVs) actively formed in response to cell activation and provides a sophisticated structural mechanism for delivery of granule-stored mediators. In this review, we highlight the application of EM techniques to recognize pools of immune mediators at vesicular compartments and to understand the complex secretory pathway within human eosinophils involved in inflammatory and allergic responses. - Highlights: • Application of EM to understand the complex secretory pathway in human eosinophils. • EM techniques reveal an active vesicular system associated with secretory granules. • Tubular vesicles are involved in the transport of granule-derived immune mediators.

HUMAN CATALASE IS IMPORTED AND ASSEMBLED IN PEROXISOMES OF SACCHAROMYCES-CEREVISIAE

NARCIS (Netherlands)

DEHOOP, MJ; HOLTMAN, WL; AB, G

To study the conservation of peroxisomal targeting signals, we have determined the intracellular localization of human peroxisomal catalase when expressed in yeast. Using immunofluorescence, differential centrifugation and immuno-electron microscopy, we show that the protein is targeted to the

Absence of E protein arrests transmissible gastroenteritis coronavirus maturation in the secretory pathway

International Nuclear Information System (INIS)

Ortego, Javier; Ceriani, Juan E.; Patino, Cristina; Plana, Juan; Enjuanes, Luis

2007-01-01

A recombinant transmissible gastroenteritis coronavirus (rTGEV) in which E gene was deleted (rTGEV-ΔE) has been engineered. This deletion mutant only grows in cells expressing E protein (E + cells) indicating that E was an essential gene for TGEV replication. Electron microscopy studies of rTGEV-ΔE infected BHK-pAPN-E - cells showed that only immature intracellular virions were assembled. These virions were non-infectious and not secreted to the extracellular medium in BHK-pAPN-E - cells. RNA and protein composition analysis by RNase-gold and immunoelectron microscopy showed that rTGEV-ΔE virions contained RNA and also all the structural TGEV proteins, except the deleted E protein. Nevertheless, full virion maturation was blocked. Studies of the rTGEV-ΔE subcellular localization by confocal and immunoelectron microscopy in infected E - cells showed that in the absence of E protein virus trafficking was arrested in the intermediate compartment. Therefore, the absence of E protein in TGEV resulted in two actions, a blockade of virus trafficking in the membranes of the secretory pathway, and prevention of full virus maturation

Localization of the AP-3 adaptor complex defines a novel endosomal exit site for lysosomal membrane proteins

NARCIS (Netherlands)

Peden, A.A.; Oorschot, V.; Hesser, B.A.; Austin, C.D.; Scheller, R.H.; Klumperman, J.

2004-01-01

The adaptor protein (AP) 3 adaptor complex has been implicated in the transport of lysosomal membrane proteins, but its precise site of action has remained controversial. Here, we show by immuno-electron microscopy that AP-3 is associated with budding profiles evolving from a tubular endosomal

The development of exo-erythrocytic schizonts of Plasmodium berghei in vitro from gamma-irradiated and non-irradiated sporozoites: a study using confocal laser scanning microscopy

International Nuclear Information System (INIS)

Sinden, E.; Couchman, A.; Suhrbier, A.; Marsh, F.; Winger, L.; Ranawaka, G.

1991-01-01

Confocal scanning laser microscopy has been used to study the distribution of antigens expressed by the liver stages of Plasmodium berghei in cultured hepatoma cells. The 3-dimensional images obtained of intact parasites clearly show complex patterns of antigen expression not apparent when using conventional IFAT or immunoelectron microscopy. A liver-stage specific antigen (Pbl 1) was shown to be confined to the parasitophorous vacuole; the vacuole has extensive diverticulae extending into the host cell. Small parasites were detected for the first time in 'mature' cultures. These did not represent a distinct population, but the 'tail' of a broad continuum of parasite sizes. Irradiated sporozoites produce a transient population of slow-growing parasites which express a very limited range of antigens de novo in the invaded hepatoma cell. A comparison of the reactivity of normal EE parasites with anti-circumsporozoite antibody and with ant-Pbl 1 suggests that the former reagent may reliably be used to identify sporozoites invading host cells, but should not be used to determine the number of parasites that successfully undergo intrahepatic development. Anti-Pbl-1 indicates on 33% of invaded sporozoites identified by anti-CSP subsequently differentiate. (author)

Expression of agrin, dystroglycan, and utrophin in normal renal tissue and in experimental glomerulopathies.

Science.gov (United States)

Raats, C J; van den Born, J; Bakker, M A; Oppers-Walgreen, B; Pisa, B J; Dijkman, H B; Assmann, K J; Berden, J H

2000-05-01

The dystrophin-glycoprotein complex, which comprises alpha- and beta-dystroglycan, sarcoglycans, and utrophin/dystrophin, links the cytoskeleton to agrin and laminin in the basal lamina in muscle and epithelial cells. Recently, agrin was identified as a major heparan sulfate proteoglycan in the glomerular basement membrane. In the present study, we found mRNA expression for agrin, dystroglycan, and utrophin in kidney cortex, isolated glomeruli, and cultured podocytes and mesangial cells. In immunofluorescence, agrin was found in the glomerular basement membrane. The antibodies against alpha- and beta-dystroglycan and utrophin revealed a granular podocyte-like staining pattern along the glomerular capillary wall. With immunoelectron microscopy, agrin was found in the glomerular basement membrane, dystroglycan was diffusely found over the entire cell surface of the podocytes, and utrophin was localized in the cytoplasm of the podocyte foot processes. In adriamycin nephropathy, a decrease in the glomerular capillary wall staining for dystroglycan was observed probably secondary to the extensive fusion of foot processes. Immunoelectron microscopy showed a different distribution pattern as compared to the normal kidney, with segmentally enhanced expression of dystroglycan at the basal side of the extensively fused podocyte foot processes. In passive Heymann nephritis we observed no changes in the staining intensity and distribution of the dystrophin-glycoprotein complex by immunofluorescence and immunoelectron microscopy. From these data, we conclude that agrin, dystroglycan, and utrophin are present in the glomerular capillary wall and their ultrastructural localization supports the concept that these molecules are involved in linking the podocyte cytoskeleton to the glomerular basement membrane.

Immunochemical and biological properties of a mouse monoclonal antibody reactive to prunus necrotic ringspot ilarvirus.

Science.gov (United States)

Aebig, J A; Jordan, R L; Lawson, R H; Hsu, H T

1987-01-01

A monoclonal antibody reacting with prunus necrotic ringspot ilarvirus was tested in immunochemical studies, neutralization of infectivity assays, and by immuno-electron microscopy. The antibody was able to detect the 27,000 Mr coat protein of prunus necrotic ringspot ilarvirus in western blots and also detected all polypeptide fragments generated after incubation of whole virus with proteolytic enzymes. In neutralization of infectivity studies, the antibody blocked virus infectivity, although it did not precipitate the antigen in agar gel Ouchterlony double diffusion tests. Immuno-electron microscopy confirmed that the antibody coats virions but does not cause clumping. The antibody may be a useful tool for investigating coat protein-dependent initiation of ilarvirus infection.

Anti-nucleosome antibodies complexed to nucleosomal antigens show anti-DNA reactivity and bind to rat glomerular basement membrane in vivo.

Science.gov (United States)

Kramers, C; Hylkema, M N; van Bruggen, M C; van de Lagemaat, R; Dijkman, H B; Assmann, K J; Smeenk, R J; Berden, J H

1994-01-01

Histones can mediate the binding of DNA and anti-DNA to the glomerular basement membrane (GBM). In ELISA histone/DNA/anti-DNA complexes are able to bind to heparan sulfate (HS), an intrinsic constituent of the GBM. We questioned whether histone containing immune complexes are able to bind to the GBM, and if so, whether the ligand in the GBM is HS. Monoclonal antibodies (mAbs) complexed to nucleosomal antigens and noncomplexed mAbs were isolated from culture supernatants of four IgG anti-nuclear mAbs. All noncomplexed mAbs showed strong anti-nucleosome reactivity in ELISA. One of them showed in addition anti-DNA reactivity in noncomplexed form. The other three mAbs only showed anti-DNA reactivity when they were complexed to nucleosomal antigens. After renal perfusion a fine granular binding of complexed mAbs to the glomerular capillary wall and activation of complement was observed in immunofluorescence, whereas noncomplexed mAbs did not bind. Immuno-electron microscopy showed binding of complexes to the whole width of the GBM. When HS in the GBM was removed by renal heparinase perfusion the binding of complexed mAb decreased, but did not disappear completely. We conclude that anti-nucleosome mAbs, which do not bind DNA, become DNA reactive once complexed to nucleosomal antigens. These complexed mAbs can bind to the GBM. The binding ligand in the GBM is partly, but not solely, HS. Binding to the GBM of immune complexes containing nucleosomal material might be an important event in the pathogenesis of lupus nephritis. Images PMID:8040312

Analysis of Polymorphic Membrane Protein Expression in Cultured Cells Identifies PmpA and PmpH of Chlamydia psittaci as Candidate Factors in Pathogenesis and Immunity to Infection.

Science.gov (United States)

Van Lent, Sarah; De Vos, Winnok H; Huot Creasy, Heather; Marques, Patricia X; Ravel, Jacques; Vanrompay, Daisy; Bavoil, Patrik; Hsia, Ru-Ching

2016-01-01

The polymorphic membrane protein (Pmp) paralogous families of Chlamydia trachomatis, Chlamydia pneumoniae and Chlamydia abortus are putative targets for Chlamydia vaccine development. To determine whether this is also the case for Pmp family members of C. psittaci, we analyzed transcription levels, protein production and localization of several Pmps of C. psittaci. Pmp expression profiles were characterized using quantitative real-time PCR (RT-qPCR), immunofluorescence (IF) and immuno-electron microscopy (IEM) under normal and stress conditions. We found that PmpA was highly produced in all inclusions as early as 12 hpi in all biological replicates. In addition, PmpA and PmpH appeared to be unusually accessible to antibody as determined by both immunofluorescence and immuno-electron microscopy. Our results suggest an important role for these Pmps in the pathogenesis of C. psittaci, and make them promising candidates in vaccine development.

Specific binding of beta-endorphin to normal human erythrocytes

Energy Technology Data Exchange (ETDEWEB)

Chenet, B.; Hollis, V. Jr.; Kang, Y.; Simpkins, C.

1986-03-05

Beta-endorphin (BE) exhibits peripheral functions which may not be mediated by interactions with receptors in the brain. Recent studies have demonstrated binding of BE to both opioid and non-opioid receptors on lymphocytes and monocytes. Abood has reported specific binding of /sup 3/H-dihydromorphine in erythrocytes. Using 5 x 10/sup -11/M /sup 125/I-beta-endorphin and 10/sup -5/M unlabeled BE, they have detected 50% specific binding to human erythrocytes. This finding is supported by results from immunoelectron microscopy using rabbit anti-BE antibody and biotinylated secondary antibody with avidin-biotin complexes horseradish peroxidase. Binding is clearly observed and is confined to only one side of the cells. Conclusions: (1) BE binding to human erythrocytes was demonstrated by radioreceptor assay and immunoelectron microscopy, and (2) BE binding sites exist on only one side of the cells.

Immunocytochemical localization of APS reductase and bisulfite reductase in three Desulfovibrio species

NARCIS (Netherlands)

Kremer, D.R.; Veenhuis, M.; Fauque, G.; Peck Jr., H.D.; LeGall, J.; Lampreia, J.; Moura, J.J.G.; Hansen, T.A.

1988-01-01

The localization of APS reductase and bisulfite reductase in Desulfovibrio gigas, D. vulgaris Hildenborough and D. thermophilus was studied by immunoelectron microscopy. Polyclonal antibodies were raised against the purified enzymes from each strain. Cells fixed with formaldehyde/glutaraldehyde were

Mechanism of lymphocytic choriomeningitis virus entry into cells.

Science.gov (United States)

Borrow, P; Oldstone, M B

1994-01-01

The path that the arenavirus lymphocytic choriomeningitis virus (LCMV) uses to enter rodent fibroblastic cell lines was dissected by infectivity and inhibition studies and immunoelectron microscopy. Lysosomotropic weak bases (chloroquine and ammonium chloride) and carboxylic ionophores (monensin and nigericin) inhibited virus entry, assessed as virus nucleoprotein expression at early times post-infection, indicating that the entry process involved a pH-dependent fusion step in intracellular vesicles. That entry occurred in vesicles rather than by direct fusion of virions with the plasma membrane was confirmed by immunoelectron microscopy. The vesicles involved were large (150-300 nm diameter), smooth-walled, and not associated with clathrin. Unlike classical phagocytosis, virus uptake in these vesicles was a microfilament-independent process, as it was not blocked by cytochalasins. LCMV entry into rodent fibroblast cell lines thus involves viropexis in large smooth-walled vesicles, followed by a pH-dependent fusion event inside the cell.

Visualization by high resolution immunoelectron microscopy of the transient receptor potential vanilloid-1 at inhibitory synapses of the mouse dentate gyrus.

Directory of Open Access Journals (Sweden)

Miren-Josune Canduela

Full Text Available We have recently shown that the transient receptor potential vanilloid type 1 (TRPV1, a non-selective cation channel in the peripheral and central nervous system, is localized at postsynaptic sites of the excitatory perforant path synapses in the hippocampal dentate molecular layer (ML. In the present work, we have studied the distribution of TRPV1 at inhibitory synapses in the ML. With this aim, a preembedding immunogold method for high resolution electron microscopy was applied to mouse hippocampus. About 30% of the inhibitory synapses in the ML are TRPV1 immunopositive, which is mostly localized perisynaptically (∼60% of total immunoparticles at postsynaptic dendritic membranes receiving symmetric synapses in the inner 1/3 of the layer. This TRPV1 pattern distribution is not observed in the ML of TRPV1 knock-out mice. These findings extend the knowledge of the subcellular localization of TRPV1 to inhibitory synapses of the dentate molecular layer where the channel, in addition to excitatory synapses, is present.

Ultrastructural immunocytochemistry of particulate fractions using polyvinyl chloride microculture wells.

Science.gov (United States)

Wray, B E; Sealock, R

1984-10-01

A method is described for immunoelectron microscopy of particulate subcellular fractions using polyvinyl chloride (soft) microculture wells as mechanical supports and reaction vessels. Appropriate quantities of particles are centrifuged onto the well bottoms, fixed and permeabilized if necessary, then labeled by standard procedures, fixed in glutaraldehyde and tannic acid, and prepared for thin section electron microscopy. The centrifugation, the fixations, and the embedment in Epon are discussed in detail.

Evidence for small intracellular vesicles in human blood phagocytes containing cytochrome b558 and the adhesion molecule CD11b/CD18

NARCIS (Netherlands)

Calafat, J.; Kuijpers, T. W.; Janssen, H.; Borregaard, N.; Verhoeven, A. J.; Roos, D.

1993-01-01

Human neutrophils contain a rapidly mobilizable pool of so-called secretory vesicles distinct from the azurophil granules and specific granules. Using human albumin as a marker for these intracellular vesicles in immuno-electron microscopy, we found that part of the cytochrome b558 in non-purified

Somatostatinoma: collision with neurofibroma and ultrastructural features.

Science.gov (United States)

Varikatt, W; Yong, J L C; Killingsworth, M C

2006-11-01

The clinical presentation, histopathology and immunoelectron microscopic features of two cases of duodenal somatostatinoma are described, one of which is a hitherto unreported example of a collision tumour with a neurofibroma. Ultrastructural morphometric immunoelectron microscopy studies revealed the presence of four types of cells in both tumours, but there was no difference in the proportions of these cells between the collision tumour and the non-collision tumour. Neurosecretory granules ranging in size from 255-815 nm were generally larger than those previously reported for somatostatinomas and somatostatin was identified in granules of all sizes across this range. Neither tumour was associated with the somatostatinoma syndrome comprising associated diabetes mellitis, steatorrhoea and cholelithiasis.

Minor displacements in the insertion site provoke major differences in the induction of antibody responses by chimeric parvovirus-like particles

DEFF Research Database (Denmark)

Rueda, P.; Hurtado, A.; del Barrio, M.

1999-01-01

inserted into these positions was well recognized by the specific monoclonal antibody C3 by immunoelectron microscopy. BALB/c mice immunized with these chimeric C3:B CPV:VLPs were able to elicit an strong neutralizing antibody response (>3 log(10) units) against poliovirus type 1 (Mahoney strain...

Estimation of the number of angiotensin II AT1 receptors in rat kidney afferent and efferent arterioles

DEFF Research Database (Denmark)

Razga, Zsolt; Nyengaard, Jens Randel

2007-01-01

of angiotensin II AT1 receptors along the length of the arterioles and per arteriole, we combined immunoelectron microscopy with stereology. RESULTS: The number of AT1 receptor molecules was significantly lower in the renin-positive smooth muscle cells (SMCs) than in the renin-negative SMCs of the afferent...

Changes in cell morphology of Listeria monocytogenesnes and Shewanella putrefaciens resulting from the action of protamine

DEFF Research Database (Denmark)

Johansen, Charlotte; Gill, T.; Gram, Lone

1996-01-01

cells. Immunoelectron microscopy of protamine-treated cells of both L. monocytogenes and S. putrefaciens showed great damage to the cell wall and condensation of the cytoplasm. Respiration of the cells was decreased due to treatment with sublethal concentrations of protamine, probably due to leakage...... or loss of cell envelope potential. It was concluded that protamine disrupted the outer surface structure and condensed the cytoplasm of sensitive cells and, in sublethal concentrations, altered membrane structures, thereby eliminating respiration......Protamine, which is an antibacterial basic peptide, was shown to alter the cell morphology of Listeria monocytogenes and Shewanella putrefaciens. Atomic force microscopy revealed that protamine smoothed the surface of cells, formed holes in the cell envelope, and caused fusion of S. putrefaciens...

Anatomical characterization of cytoglobin and neuroglobin mRNA and protein expression in the mouse brain

DEFF Research Database (Denmark)

Hundahl, Christian Ansgar; Allen, Gregg C; Hannibal, Jens

2010-01-01

The present study aimed at characterizing the anatomical and subcellular localization of cytoglobin (Cygb) and neuroglobin (Ngb) in the mouse brain by use of in situ hybridisation, immunohistochemistry and immunoelectron microscopy. Cygb and Ngb were only found in distinct brain areas and often i...... for Cygb and involvement in sleep-wake cycling for Cygb and Ngb....

Innovative Strategies for Clinical Microscopy Instruction: Virtual Versus Light Microscopy.

Science.gov (United States)

McDaniel, M Jane; Russell, Gregory B; Crandall, Sonia J

2018-06-01

The purpose of the study was to compare virtual microscopy with light microscopy to determine differences in learning outcomes and learner attitudes in teaching clinical microscopy to physician assistant (PA) students. A prospective, randomized, crossover design study was conducted with a convenience sample of 67 first-year PA students randomized to 2 groups. One group used light microscopes to find microscopic structures, whereas the other group used instructor-directed video streaming of microscopic elements. At the midpoint of the study, the groups switched instructional strategies. Learning outcomes were assessed via posttest after each section of the study, with comparison of final practical examination results to previous cohorts. Attitudes about the 2 educational strategies were assessed through a postcourse questionnaire with a Likert scale. Analysis of the first posttest demonstrated that students in the video-streamed group had significantly better learning outcomes than those in the light microscopy group (P = .004; Cohen's d = 0.74). Analysis of the posttest after crossover showed no differences between the 2 groups (P = .48). Between the 2 posttests, students first assigned to the light microscopy group scored a 6.6 mean point increase (±10.4 SD; p = .0011), whereas students first assigned to the virtual microscopy group scored a 1.3 mean point increase (±7.1 SD; p = .29). The light microscopy group improved more than the virtual microscopy group (P = .019). Analysis of practical examination data revealed higher scores for the study group compared with 5 previous cohorts of first-year students (P virtual microscopy to traditional light microscopy. Virtual microscopy is an effective educational strategy, and students prefer this method when learning to interpret images of clinical specimens.

Periostin is an extracellular matrix protein required for eruption of incisors in mice

International Nuclear Information System (INIS)

Kii, Isao; Amizuka, Norio; Minqi, Li; Kitajima, Satoshi; Saga, Yumiko; Kudo, Akira

2006-01-01

A characteristic tooth of rodents, the incisor continuously grows throughout life by the constant formation of dentin and enamel. Continuous eruption of the incisor is accompanied with formation of shear zone, in which the periodontal ligament is remodeled. Although the shear zone plays a role in the remodeling, its molecular biological aspect is barely understood. Here, we show that periostin is essential for formation of the shear zone. Periostin -/- mice showed an eruption disturbance of incisors. Histological observation revealed that deletion of periostin led to disappearance of the shear zone. Electron microscopy revealed that the disappearance of the shear zone resulted from a failure in digestion of collagen fibers in the periostin -/- mice. Furthermore, immunohistochemical analysis using anti-periostin antibodies demonstrated the restricted localization of periostin protein in the shear zone. Periostin is an extracellular matrix protein, and immunoelectron microscopy showed a close association of periostin with collagen fibrils in vivo. These results suggest that periostin functions in the remodeling of collagen matrix in the shear zone

Transmission and scanning electron microscopy study of the characteristics and morphology of pericytes and novel desmin-immunopositive perivascular cells before and after castration in rat anterior pituitary gland.

Science.gov (United States)

Jindatip, Depicha; Fujiwara, Ken; Kouki, Tom; Yashiro, Takashi

2012-09-01

Pericytes are perivascular cells associated with microcirculation. Typically, they are localized close to the capillary wall, underneath the basement membrane, and have sparse cytoplasm and poorly developed cell organelles. However, the specific properties of pericytes vary by organ and the conditions within organs. We recently demonstrated that pericytes in rat anterior pituitary gland produce type I and III collagens. The present study attempted to determine the morphological characteristics of these pituitary pericytes. Castrated rats were used as a model of hormonal and vascular changes in the gland. Pericytes, as determined by desmin immunohistochemistry, were more numerous and stained more intensely in castrated rats. Transmission electron microscopy revealed that pituitary pericytes displayed the typical characteristics of pericytes. In pituitary sections from castrated rats, the Golgi apparatus of pericytes was well developed and the rough endoplasmic reticulum was elongated. Additionally, scanning electron microscopy revealed four pericyte shapes: oval, elongate, triangular, and multiangular. As compared with normal rats, the proportion of oval pericytes was lower, and the proportions of the other three shapes were higher, in castrated rats. These results suggest that pericytes change their fine structure and cell shape in response to hormonal and vascular changes in the anterior pituitary gland. In addition, a novel type of perivascular cell was found by desmin immunoelectron microscopy. The morphological properties of these cells were dissimilar to those of pericytes. The cells were localized in the perivascular space, had no basement membrane, and contained dilated rough endoplasmic reticulum. This new cell type will require further study of its origin and characteristics.

Dipeptidyl peptidase IV is sorted to the secretory granules in pancreatic islet A-cells

DEFF Research Database (Denmark)

Poulsen, Mona Dam; Hansen, Gert Helge; Dabelsteen, Erik

1993-01-01

Dipeptidyl peptidase IV (DP IV:EC 3.4.14.5) was localized in endocrine cells of pig pancreas by immunohistochemical and enzyme histochemical methods. Immunolight microscopy with both monoclonal and polyclonal antibodies demonstrated DP IV immunoreactivity in cells located in the peripheral part...... of the islets of Langerhans. The antigen is enzymatically active, as shown by enzyme histochemical analysis with a synthetic DP IV substrate. By immunoelectron microscopy (immunogold labeling), the labeling of DP IV in the islets was associated with the secretory granules of the A-cells, as identified by double...... labeling using a monoclonal glucagon antibody as the second primary antibody. These results show that DP IV is sorted to secretory granules in the pig pancreatic islet A-cells. Furthermore, this secretory granule enzyme, as opposed to intestinal brush border DP IV, is suggested to be a soluble protein...

Microsphere imaging with confocal microscopy and two photon microscopy

International Nuclear Information System (INIS)

Chun, Hyung Su; An, Kyung Won; Lee, Jai Hyung

2002-01-01

We have acquired images of polystyrene and fused-silica microsphere by using conventional optical microscopy, confocal microscopy and two-photon microscopy, and performed comparative analysis of these images. Different from conventional optical microscopy, confocal and two-photon microscopy had good optical sectioning capability. In addition, confocal microscopy and two-photon microscopy had better lateral resolution than conventional optical microscopy. These results are attributed to confocality and nonlinearity of confocal microscopy and two photon microscopy, respectively.

Penetration of silver nanoparticles into porcine skin ex vivo using fluorescence lifetime imaging microscopy, Raman microscopy, and surface-enhanced Raman scattering microscopy.

Science.gov (United States)

Zhu, Yongjian; Choe, Chun-Sik; Ahlberg, Sebastian; Meinke, Martina C; Alexiev, Ulrike; Lademann, Juergen; Darvin, Maxim E

2015-05-01

In order to investigate the penetration depth of silver nanoparticles (Ag NPs) inside the skin, porcine ears treated with Ag NPs are measured by two-photon tomography with a fluorescence lifetime imaging microscopy (TPT-FLIM) technique, confocal Raman microscopy (CRM), and surface-enhanced Raman scattering (SERS) microscopy. Ag NPs are coated with poly-N-vinylpyrrolidone and dispersed in pure water solutions. After the application of Ag NPs, porcine ears are stored in the incubator for 24 h at a temperature of 37°C. The TPT-FLIM measurement results show a dramatic decrease of the Ag NPs' signal intensity from the skin surface to a depth of 4 μm. Below 4 μm, the Ag NPs' signal continues to decline, having completely disappeared at 12 to 14 μm depth. CRM shows that the penetration depth of Ag NPs is 11.1 ± 2.1 μm. The penetration depth measured with a highly sensitive SERS microscopy reaches 15.6 ± 8.3 μm. Several results obtained with SERS show that the penetration depth of Ag NPs can exceed the stratum corneum (SC) thickness, which can be explained by both penetration of trace amounts of Ag NPs through the SC barrier and by the measurements inside the hair follicle, which cannot be excluded in the experiment.

Probing cytotoxicity of nanoparticles and organic compounds using scanning proton microscopy, scanning electron microscopy and fluorescence microscopy

Energy Technology Data Exchange (ETDEWEB)

Tong Yongpeng [Institute of Nuclear Techniques, Shenzhen University, Nanhai Avenue 3688, Shenzhen 518060 (China)], E-mail: yongpengt@yahoo.com.cn; Li Changming [School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore 637457 (Singapore); Liang Feng [Institute Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025 (China); Chen Jianmin [Shenzhen Municipal Hospital for Chronic Disease Control and Prevention, Guangdong 518020 (China); Zhang Hong; Liu Guoqing; Sun Huibin [Institute of Nuclear Techniques, Shenzhen University, Nanhai Avenue 3688, Shenzhen 518060 (China); Luong, John H.T. [Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec, H4P 2R2 (Canada)

2008-12-15

Scanning proton microscopy, scanning electron microscopy (SEM) and fluorescence microscopy have been used to probe the cytotoxicity effect of benzo[a]pyrene (BaP), ethidium bromide (EB) and nanoparticles (ZnO, Al{sub 2}O{sub 3} and TiO{sub 2}) on a T lymphoblastic leukemia Jurkat cell line. The increased calcium ion (from CaCl{sub 2}) in the culture medium stimulated the accumulation of BaP and EB inside the cell, leading to cell death. ZnO, Al{sub 2}O{sub 3} and TiO{sub 2} nanoparticles, however, showed a protective effect against these two organic compounds. Such inorganic nanoparticles complexed with BaP or EB which became less toxic to the cell. Fe{sub 2}O{sub 3} nanoparticles as an insoluble particle model scavenged by macrophage were investigated in rats. They were scavenged out of the lung tissue about 48 h after infection. This result suggest that some insoluble inorganic nanoparticles of PM (particulate matters) showed protective effects on organic toxins induced acute toxic effects as they can be scavenged by macrophage cells. Whereas, some inorganic ions such as calcium ion in PM may help environmental organic toxins to penetrate cell membrane and induce higher toxic effect.

Ultrastructural and biochemical evidence for the presence of mature steroidogenic acute regulatory protein (StAR) in the cytoplasm of human luteal cells.

Science.gov (United States)

Sierralta, Walter D; Kohen, Paulina; Castro, Olga; Muñoz, Alex; Strauss, Jerome F; Devoto, Luigi

2005-10-20

The distribution of the steroidogenic acute regulatory protein (StAR) inside thecal and granulosa-lutein cells of human corpus luteum (CL) was assessed by immunoelectron microscopy. We found greater levels of StAR immunolabeling in steroidogenic cells from early- and mid-than in late luteal phase CL and lower levels in cells from women treated with a GnRH antagonist in the mid-luteal phase. Immunoelectron microscopy revealed significant levels of StAR antigen in the mitochondria and in the cytoplasm of luteal cells. The 30 kDa mature StAR protein was present in both mitochondria and cytosol (post-mitochondrial) fractions from homogenates of CL at different ages, whereas cytochrome c and mitochondrial HSP70 were detected only in the mitochondrial fraction. Therefore, we hypothesized that either appreciable processing of StAR 37 kDa pre-protein occurs outside the mitochondria, or mature StAR protein is selectively released into the cytoplasm after mitochondrial processing. The presence of mature StAR in the cytoplasm is consonant with the notion that StAR acts on the outer mitochondrial membrane to effect sterol import, and that StAR may interact with other cytoplasmic proteins involved in cholesterol metabolism, including hormone sensitive lipase.

Dysprosium disilicide nanostructures on silicon(001) studied by scanning tunneling microscopy and transmission electron microscopy

International Nuclear Information System (INIS)

Ye Gangfeng; Nogami, Jun; Crimp, Martin A.

2006-01-01

The microstructure of self-assembled dysprosium silicide nanostructures on silicon(001) has been studied by scanning tunneling microscopy and transmission electron microscopy. The studies focused on nanostructures that involve multiple atomic layers of the silicide. Cross-sectional high resolution transmission electron microscopy images and fast Fourier transform analysis showed that both hexagonal and orthorhombic/tetragonal silicide phases were present. Both the magnitude and the anisotropy of lattice mismatch between the silicide and the substrate play roles in the morphology and epitaxial growth of the nanostructures formed

Changes in Subcellular Distribution of n-Octanoyl or n-Decanoyl Ghrelin in Ghrelin-Producing Cells

OpenAIRE

Nishi, Yoshihiro; Mifune, Hiroharu; Yabuki, Akira; Tajiri, Yuji; Hirata, Rumiko; Tanaka, Eiichiro; Hosoda, Hiroshi; Kangawa, Kenji; Kojima, Masayasu

2013-01-01

Background: The enzyme ghrelin O-acyltransferase (GOAT) catalyzes the acylation of ghrelin. The molecular form of GOAT, together with its reaction in vitro, has been reported previously. However, the sub-cellular processes governing the acylation of ghrelin remain to be elucidated.Methods: Double immunoelectron microscopy was used to examine changes in the relative proportions of secretory granules containing n-octanoyl ghrelin (C8-ghrelin) or n-decanoyl ghrelin (C10-ghrelin) in ghrelin-pro...

Atomic force microscopy and confocal laser scanning microscopy on the cytoskeleton of permeabilised and embedded cells

International Nuclear Information System (INIS)

Meller, Karl; Theiss, Carsten

2006-01-01

We describe a technical method of cell permeabilisation and embedding to study the organisation and distribution of intracellular proteins with aid of atomic force microscopy and confocal laser scanning microscopy in identical areas. While confocal laser scanning microscopy is useful for the identification of certain proteins subsequent labelling with markers or antibodies, atomic force microscopy allows the observation of macromolecular structures in fixed and living cells. To demonstrate the field of application of this preparatory technique, cells were permeabilised, fixed, and the actin cytoskeleton was stained with phalloidin-rhodamine. Confocal laser scanning microscopy was used to show the organisation of these microfilaments, e.g. geodesic dome structures. Thereafter, cells were embedded in Durcupan water-soluble resin, followed by UV-polymerisation of resin at 4 o C. This procedure allowed intracellular visualisation of the cell nucleus or cytoskeletal elements by atomic force microscopy, for instance to analyse the globular organisation of actin filaments. Therefore, this method offers a great potential to combine both microscopy techniques in order to understand and interpret intracellular protein relations, for example, the biochemical and morphological interaction of the cytoskeleton

Correlative Stochastic Optical Reconstruction Microscopy and Electron Microscopy

Science.gov (United States)

Kim, Doory; Deerinck, Thomas J.; Sigal, Yaron M.; Babcock, Hazen P.; Ellisman, Mark H.; Zhuang, Xiaowei

2015-01-01

Correlative fluorescence light microscopy and electron microscopy allows the imaging of spatial distributions of specific biomolecules in the context of cellular ultrastructure. Recent development of super-resolution fluorescence microscopy allows the location of molecules to be determined with nanometer-scale spatial resolution. However, correlative super-resolution fluorescence microscopy and electron microscopy (EM) still remains challenging because the optimal specimen preparation and imaging conditions for super-resolution fluorescence microscopy and EM are often not compatible. Here, we have developed several experiment protocols for correlative stochastic optical reconstruction microscopy (STORM) and EM methods, both for un-embedded samples by applying EM-specific sample preparations after STORM imaging and for embedded and sectioned samples by optimizing the fluorescence under EM fixation, staining and embedding conditions. We demonstrated these methods using a variety of cellular targets. PMID:25874453

Nano-contact microscopy of supracrystals

Directory of Open Access Journals (Sweden)

Adam Sweetman

2015-05-01

Full Text Available Background: Highly ordered three-dimensional colloidal crystals (supracrystals comprised of 7.4 nm diameter Au nanocrystals (with a 5% size dispersion have been imaged and analysed using a combination of scanning tunnelling microscopy and dynamic force microscopy.Results: By exploring the evolution of both the force and tunnel current with respect to tip–sample separation, we arrive at the surprising finding that single nanocrystal resolution is readily obtained in tunnelling microscopy images acquired more than 1 nm into the repulsive (i.e., positive force regime of the probe–nanocrystal interaction potential. Constant height force microscopy has been used to map tip–sample interactions in this regime, revealing inhomogeneities which arise from the convolution of the tip structure with the ligand distribution at the nanocrystal surface.Conclusion: Our combined STM–AFM measurements show that the contrast mechanism underpinning high resolution imaging of nanoparticle supracrystals involves a form of nanoscale contact imaging, rather than the through-vacuum tunnelling which underpins traditional tunnelling microscopy and spectroscopy.

Scanning Capacitance Microscopy | Materials Science | NREL

Science.gov (United States)

obtained using scanning capacitance microscopy. Top Right: Image of p-type and n-type material, obtained 'fingers' of light-colored n-type material on a yellow and blue background representing p-type material material, obtained using scanning capacitance microscopy, in a sample semiconductor device; the image shows

Fluorescence microscopy.

Science.gov (United States)

Sanderson, Michael J; Smith, Ian; Parker, Ian; Bootman, Martin D

2014-10-01

Fluorescence microscopy is a major tool with which to monitor cell physiology. Although the concepts of fluorescence and its optical separation using filters remain similar, microscope design varies with the aim of increasing image contrast and spatial resolution. The basics of wide-field microscopy are outlined to emphasize the selection, advantages, and correct use of laser scanning confocal microscopy, two-photon microscopy, scanning disk confocal microscopy, total internal reflection, and super-resolution microscopy. In addition, the principles of how these microscopes form images are reviewed to appreciate their capabilities, limitations, and constraints for operation. © 2014 Cold Spring Harbor Laboratory Press.

Immunolocalization of an enterotoxic glycoprotein exoantigen on the secretory organelles of Cryptosporidium parvum sporozoites

Directory of Open Access Journals (Sweden)

El-Shewy K.A.

2004-06-01

Full Text Available In this study, the fine ultrastructures of the secretory organelles of C. parvum sporozoites were demonstrated using transmission electron microscopy (TEM. Meanwhile, a previously identified enterotoxic 18-20 kDa copro-antigen (18-20 kDa CCA, associated with cryptosporidiosis in both human and calves, was isolated and immunolocalized on C. parvum sporozoites. Using immunoelectron microscopy and anti-18-20 kDa monospecific antibody demonstrated marked existence of the 18-20 kDa CCA on the apical organelles and at the trilaminar pellicles. An anterior extrusion of this protein was demonstrated around the excysted and released sporozoites. However, non excysted sporozoites did not show this protein. Affinity blotting, with biotinylated jacalin, demonstrated the O-linked oligosaccharide moiety of this protein. The potential role of this protein in the host cell invasion and/or gliding motility remains unelucidated. However, its enterotoxicity, location and secretory nature suggest that it may be a target for neutralization or invasion inhibition of Cryptosporidium.

Autophagy-related Atg8 localizes to the apicoplast of the human malaria parasite Plasmodium falciparum.

Directory of Open Access Journals (Sweden)

Kei Kitamura

Full Text Available Autophagy is a membrane-mediated degradation process, which is governed by sequential functions of Atg proteins. Although Atg proteins are highly conserved in eukaryotes, protozoa possess only a partial set of Atg proteins. Nonetheless, almost all protozoa have the complete factors belonging to the Atg8 conjugation system, namely, Atg3, Atg4, Atg7, and Atg8. Here, we report the biochemical properties and subcellular localization of the Atg8 protein of the human malaria parasite Plasmodium falciparum (PfAtg8. PfAtg8 is expressed during intra-erythrocytic development and associates with membranes likely as a lipid-conjugated form. Fluorescence microscopy and immunoelectron microscopy show that PfAtg8 localizes to the apicoplast, a four membrane-bound non-photosynthetic plastid. Autophagosome-like structures are not observed in the erythrocytic stages. These data suggest that, although Plasmodium parasites have lost most Atg proteins during evolution, they use the Atg8 conjugation system for the unique organelle, the apicoplast.

Nanoparticle sizing: a comparative study using atomic force microscopy, transmission electron microscopy, and ferromagnetic resonance

International Nuclear Information System (INIS)

Lacava, L.M.; Lacava, B.M.; Azevedo, R.B.; Lacava, Z.G.M.; Buske, N.; Tronconi, A.L.; Morais, P.C.

2001-01-01

Atomic force microscopy (AFM), transmission electron microscopy (TEM), and ferromagnetic resonance (FMR) were used to unfold the nanoparticle size of a ferrofluid sample. Compared to TEM, the AFM method showed a nanoparticle diameter (D m ) reduction of 20% and standard deviation (σ) increase of 15%. The differences in D m and σ were associated with the AFM tip and the nanoparticle concentration on the substrate

[Application of immunologic methods to the analysis of bio-leaching bacteria].

Science.gov (United States)

Coto, O; Fernández, A I; León, T; Rodríguez, D

1994-09-01

Pure cultures of Thiobacillus ferrooxidans and mixed cultures of Thiobacillus ferrooxidans and Leptospirillum ferrooxidans isolated from the Matahambre mine (Cuba) were used to fit immunodiffusion and immunoelectron microscopy to the study of iron oxidizing bacteria. The possibilities, advantages and limits of those techniques have been studied from both the identification and the serological characterization points of view. Finally, the efficiency of these methods was tested by applying them to the identification of microorganisms from acidic waters from the mine.

X-ray microscopy of human malaria

International Nuclear Information System (INIS)

Magowan, C.; Brown, J.T.; Mohandas, N.; Meyer-Ilse, W.

1997-01-01

Associations between intracellular organisms and host cells are complex and particularly difficult to examine. X-ray microscopy provides transmission images of subcellular structures in intact cells at resolutions superior to available methodologies. The spatial resolution is 50-60nm with a 1 micron depth of focus, superior to anything achievable with light microscopy. Image contrast is generated by differences in photoelectric absorption by the atoms in different areas (i.e. subcellular structures) throughout the full thickness of the sample. Absorption due to carbon dominates among all the elements in the sample at 2.4 nm x-ray wavelength. Thus images show features or structures, in a way not usually seen by other types of microscopy. The authors used soft x-ray microscopy to investigate structural development of Plasmodium falciparum malaria parasites in normal and genetically abnormal erythrocytes, and in infected erythrocytes treated with compounds that have anti-malarial effects. X-ray microscopy showed newly elaborated structures in the cytosol of unstained, intact erythrocytes, redistribution of mass (carbon) in infected erythrocytes, and aberrant parasite morphology. Better understanding of the process of intracellular parasite maturation and the interactions between the parasite and its host erythrocyte can help define new approaches to the control of this deadly disease

X-ray microscopy of human malaria

Energy Technology Data Exchange (ETDEWEB)

Magowan, C.; Brown, J.T.; Mohandas, N.; Meyer-Ilse, W. [Ernest Orlando Lawrence Berkeley National Lab., CA (United States)

1997-04-01

Associations between intracellular organisms and host cells are complex and particularly difficult to examine. X-ray microscopy provides transmission images of subcellular structures in intact cells at resolutions superior to available methodologies. The spatial resolution is 50-60nm with a 1 micron depth of focus, superior to anything achievable with light microscopy. Image contrast is generated by differences in photoelectric absorption by the atoms in different areas (i.e. subcellular structures) throughout the full thickness of the sample. Absorption due to carbon dominates among all the elements in the sample at 2.4 nm x-ray wavelength. Thus images show features or structures, in a way not usually seen by other types of microscopy. The authors used soft x-ray microscopy to investigate structural development of Plasmodium falciparum malaria parasites in normal and genetically abnormal erythrocytes, and in infected erythrocytes treated with compounds that have anti-malarial effects. X-ray microscopy showed newly elaborated structures in the cytosol of unstained, intact erythrocytes, redistribution of mass (carbon) in infected erythrocytes, and aberrant parasite morphology. Better understanding of the process of intracellular parasite maturation and the interactions between the parasite and its host erythrocyte can help define new approaches to the control of this deadly disease.

Cell reactions with biomaterials: the microscopies

Directory of Open Access Journals (Sweden)

Curtis A. S.G.

2001-01-01

Full Text Available The methods and results of optical microscopy that can be used to observe cell reactions to biomaterials are Interference Reflection Microscopy (IRM, Total Internal Reflection Fluorescence Microscopy (TIRFM, Surface Plasmon Resonance Microscopy (SPRM and Forster Resonance Energy Transfer Microscopy (FRETM and Standing Wave Fluorescence Microscopy. The last three are new developments, which have not yet been fully perfected. TIRFM and SPRM are evanescent wave methods. The physics of these methods depend upon optical phenomena at interfaces. All these methods give information on the dimensions of the gap between cell and the substratum to which it is adhering and thus are especially suited to work with biomaterials. IRM and FRETM can be used on opaque surfaces though image interpretation is especially difficult for IRM on a reflecting opaque surface. These methods are compared with several electron microscopical methods for studying cell adhesion to substrata. These methods all yield fairly consistent results and show that the cell to substratum distance on many materials is in the range 5 to 30 nm. The area of contact relative to the total projected area of the cell may vary from a few per cent to close to 100% depending on the cell type and substratum. These methods show that those discrete contact areas well known as focal contacts are frequently present. The results of FRETM suggest that the separation from the substratum even in a focal contact is about 5 nm.

Non-mineralized fibrocartilage shows the lowest elastic modulus in the rabbit supraspinatus tendon insertion: measurement with scanning acoustic microscopy.

Science.gov (United States)

Sano, Hirotaka; Saijo, Yoshifumi; Kokubun, Shoichi

2006-01-01

The acoustic properties of rabbit supraspinatus tendon insertions were measured by scanning acoustic microscopy. After cutting parallel to the supraspinatus tendon fibers, specimens were fixed with 10% neutralized formalin, embedded in paraffin, and sectioned. Both the sound speed and the attenuation constant were measured at the insertion site. The 2-dimensional distribution of the sound speed and that of the attenuation constant were displayed with color-coded scales. The acoustic properties reflected both the histologic architecture and the collagen type. In the tendon proper and the non-mineralized fibrocartilage, the sound speed and attenuation constant gradually decreased as the predominant collagen type changed from I to II. In the mineralized fibrocartilage, they increased markedly with the mineralization of the fibrocartilaginous tissue. These results indicate that the non-mineralized fibrocartilage shows the lowest elastic modulus among 4 zones at the insertion site, which could be interpreted as an adaptation to various types of biomechanical stress.

Correlated Light Microscopy and Electron Microscopy

NARCIS (Netherlands)

Sjollema, Klaas A.; Schnell, Ulrike; Kuipers, Jeroen; Kalicharan, Ruby; Giepmans, Ben N. G.; MullerReichert, T; Verkade, P

2012-01-01

Understanding where, when, and how biomolecules (inter)act is crucial to uncover fundamental mechanisms in cell biology. Recent developments in fluorescence light microscopy (FLM) allow protein imaging in living cells and at the near molecular level. However, fluorescence microscopy only reveals

The major basement membrane components localize to the chondrocyte pericellular matrix--a cartilage basement membrane equivalent?

DEFF Research Database (Denmark)

Kvist, Alexander J.; Nyström, Alexander; Hultenby, Kjell

2007-01-01

In this study, we demonstrate that articular cartilage chondrocytes are surrounded by the defining basement membrane proteins laminin, collagen type IV, nidogen and perlecan, and suggest that these form the functional equivalent of a basement membrane. We found by real-time PCR that mouse...... chondrocytes express these four cardinal components of basement membranes and demonstrated by immunohistochemistry that the proteins are present in bovine and mouse cartilage tissues and are deposited in a thin pericellular structure. Immunoelectron microscopy confirmed high laminin concentration...... becomes less distinct, especially in areas of obvious mechanical attrition. Interestingly, individual laminin subunits were located in different zones of the cartilage, with laminin alpha1 showing preferential localization around a select population of superficial layer chondrocytes. We propose...

Analysis of incomplete excisions of basal-cell carcinomas after breadloaf microscopy compared with 3D-microscopy: a prospective randomized and blinded study.

Science.gov (United States)

Boehringer, Alexandra; Adam, Patrick; Schnabl, Saskia; Häfner, Hans-Martin; Breuninger, Helmut

2015-08-01

Basal-cell carcinomas may show irregular, asymmetric subclinical growth. This study analyzed the efficacy of 'breadloaf' microscopy (serial sectioning) and three-dimensional (3D) microscopy in detecting positive tumor margins. Two hundred eighty-three (283) tumors (51.2%) were put into the breadloaf microscopy group; 270 tumors (48.8%) into the 3D microscopy group. The position of any detected tumor outgrowths was identified in clock face fashion. The time required for cutting and embedding the specimens and the examination of the microscopic slides was measured. Patient/tumor characteristics and surgical margins did not differ significantly. Tumor outgrowths at the excision margin were found in 62 of 283 cases (21.9%) in the breadloaf microscopy group and in 115 of 270 cases (42.6%) in the 3D microscopy group, constituting a highly significant difference (p < 0.001). This difference held true with incomplete excision of fibrosing (infiltrative/sclerosing/morpheaform) tumors [32.9% in the breadloaf microscopy group and 57.5% in the 3D microscopy group (p = 0.003)] and also with solid (nodular) tumors [16.1 and 34.2%, respectively (p < 0.001)]. The mean overall examination time required showed no important difference. In summary, for detection of tumor outgrowths, 3D microscopy has almost twice the sensitivity of breadloaf microscopy, particularly in the situation of aggressive/infiltrative carcinomas. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Dictionary of Microscopy

Science.gov (United States)

Heath, Julian

2005-10-01

The past decade has seen huge advances in the application of microscopy in all areas of science. This welcome development in microscopy has been paralleled by an expansion of the vocabulary of technical terms used in microscopy: terms have been coined for new instruments and techniques and, as microscopes reach even higher resolution, the use of terms that relate to the optical and physical principles underpinning microscopy is now commonplace. The Dictionary of Microscopy was compiled to meet this challenge and provides concise definitions of over 2,500 terms used in the fields of light microscopy, electron microscopy, scanning probe microscopy, x-ray microscopy and related techniques. Written by Dr Julian P. Heath, Editor of Microscopy and Analysis, the dictionary is intended to provide easy navigation through the microscopy terminology and to be a first point of reference for definitions of new and established terms. The Dictionary of Microscopy is an essential, accessible resource for: students who are new to the field and are learning about microscopes equipment purchasers who want an explanation of the terms used in manufacturers' literature scientists who are considering using a new microscopical technique experienced microscopists as an aide mémoire or quick source of reference librarians, the press and marketing personnel who require definitions for technical reports.

Observation of multicellular spinning behavior of Proteus mirabilis by atomic force microscopy and multifunctional microscopy.

Science.gov (United States)

Liu, Yanxia; Deng, Yuanxin; Luo, Shuxiu; Deng, Yu; Guo, Linming; Xu, Weiwei; Liu, Lei; Liu, Junkang

2014-01-01

This study aimed to observe the multicellular spinning behavior of Proteus mirabilis by atomic force microscopy (AFM) and multifunctional microscopy in order to understand the mechanism underlying this spinning movement and its biological significance. Multifunctional microscopy with charge-coupled device (CCD) and real-time AFM showed changes in cell structure and shape of P. mirabilis during multicellular spinning movement. Specifically, the morphological characteristics of P. mirabilis, multicellular spinning dynamics, and unique movement were observed. Our findings indicate that the multicellular spinning behavior of P. mirabilis may be used to collect nutrients, perform colonization, and squeeze out competitors. The movement characteristics of P. mirabilis are vital to the organism's biological adaptability to the surrounding environment. Copyright © 2013 Elsevier Ltd. All rights reserved.

Heavy-ion microscopy

International Nuclear Information System (INIS)

Kraft, G.; Yang, T.C.H.; Richards, T.; Tobias, C.A.

1980-01-01

This chapter briefly describes the techniques of optical microscopy, scanning and transmission electron microscopy, soft x-ray microscopy and compares these latter techniques with heavy-ion microscopy. The resolution obtained with these various types of microscopy are compared and the influence of the etching procedure on total resolution is discussed. Several micrographs of mammalian cells are included

Nonlinear multicontrast microscopy of hematoxylin-and-eosin-stained histological sections

Science.gov (United States)

Tuer, Adam; Tokarz, Danielle; Prent, Nicole; Cisek, Richard; Alami, Jennifer; Dumont, Daniel J.; Bakueva, Ludmila; Rowlands, John; Barzda, Virginijus

2010-03-01

Imaging hematoxylin-and-eosin-stained cancerous histological sections with multicontrast nonlinear excitation fluorescence, second- and third-harmonic generation (THG) microscopy reveals cellular structures with extremely high image contrast. Absorption and fluorescence spectroscopy together with second hyperpolarizability measurements of the dyes shows that strong THG appears due to neutral hemalum aggregation and is subsequently enhanced by interaction with eosin. Additionally, fluorescence lifetime imaging microscopy reveals eosin fluorescence quenching by hemalums, showing better suitability of only eosin staining for fluorescence microscopy. Multicontrast nonlinear microscopy has the potential to differentiate between cancerous and healthy tissue at a single cell level.

Electron Microscopy of Ebola Virus-Infected Cells.

Science.gov (United States)

Noda, Takeshi

2017-01-01

Ebola virus (EBOV) replicates in host cells, where both viral and cellular components show morphological changes during the process of viral replication from entry to budding. These steps in the replication cycle can be studied using electron microscopy (EM), including transmission electron microscopy (TEM) and scanning electron microscopy (SEM), which is one of the most useful methods for visualizing EBOV particles and EBOV-infected cells at the ultrastructural level. This chapter describes conventional methods for EM sample preparation of cultured cells infected with EBOV.

Fibronectin non-amyloid glomerulopathy.

Science.gov (United States)

Yong, Jim L; Killingsworth, Murray C; Spicer, S Timothy; Wu, Xiao-Juan

2009-11-20

A 41-year-old Burmese man presented with nephrotic syndrome, a creatinine level of 150 micromol/L and limited clinical history. His renal biopsy demonstrated glomerulopathy with large eosinophilic deposits in the mesangium and capillary loops that were negative for Congo red, slightly positive for periodic acid-Schiff and blue with Masson trichrome stain. Immunofluorescence microscopy with a routine antibody panel was unhelpful. Electron microscopy demonstrated extensive, moderately electron-dense deposits in the subendothelial space, subepithelial space and mesangium that could be differentiated from adjacent basement membrane by their increased electron density. The deposits contained finely granular material and occasional filaments with variable diameter ranging from 9-16 nm. Fibronectin glomerulopathy was suspected from anti-fibronectin immunohistochemistry that showed positive staining of thickened capillary loops. This staining was subsequently confirmed by immunoelectron microscopy demonstrating the presence of cellular fibronectin (cFN) in deposits. Significantly, deposition of fibronectin appeared to have occurred in the absence of thickening or folding of the adjacent basement membrane. The prominent mesangial location of deposits containing a cFN isotype may indicate that retention of local fibronectin produced in the mesangium has contributed to this pathology.

Characterization of konjac glucomannan-ethyl cellulose film formation via microscopy.

Science.gov (United States)

Xiao, Man; Wan, Li; Corke, Harold; Yan, Wenli; Ni, Xuewen; Fang, Yapeng; Jiang, Fatang

2016-04-01

Konjac glucomannan-ethyl cellulose (KGM-EC, 7:3, w/w) blended film shows good mechanical and moisture resistance properties. To better understand the basis for the KGM-EC film formation, optical microscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM) were used to observe the formation of the film from emulsion. Optical microscopy images showed that EC oil droplets were homogeneously dispersed in KGM water phase without obviously coalescence throughout the entire drying process. SEM images showed the surface and cross-sectional structures of samples maintained continuous and homogeneous appearance from the emulsion to dried film. AFM images indicated that KGM molecules entangled EC molecules in the emulsion. Interactions between KGM and EC improved the stability of KGM-EC emulsion, and contributed to uniformed structures of film formation. Based on these output information, a schematic model was built to elucidate KGM-EC film-forming process. Copyright © 2015 Elsevier B.V. All rights reserved.

Second harmonic generation microscopy

DEFF Research Database (Denmark)

Brüggemann, Dagmar Adeline; Brewer, Jonathan R.; Risbo, Jens

2010-01-01

Myofibers and collagen show non-linear optical properties enabling imaging using second harmonic generation (SHG) microscopy. The technique is evaluated for use as a tool for real-time studies of thermally induced changes in thin samples of unfixed and unstained pork. The forward and the backward...... scattered SHG light reveal complementary features of the structures of myofibers and collagen fibers. Upon heating the myofibers show no structural changes before reaching a temperature of 53 °C. At this temperature the SHG signal becomes extinct. The extinction of the SHG at 53 °C coincides with a low......-temperature endotherm peak observable in the differential scanning calorimetry (DSC) thermograms. DSC analysis of epimysium, the connective tissue layer that enfold skeletal muscles, produces one large endotherm starting at 57 °C and peaking at 59.5 °C. SHG microscopy of collagen fibers reveals a variability of thermal...

Nanodiamonds as multi-purpose labels for microscopy.

Science.gov (United States)

Hemelaar, S R; de Boer, P; Chipaux, M; Zuidema, W; Hamoh, T; Martinez, F Perona; Nagl, A; Hoogenboom, J P; Giepmans, B N G; Schirhagl, R

2017-04-07

Nanodiamonds containing fluorescent nitrogen-vacancy centers are increasingly attracting interest for use as a probe in biological microscopy. This interest stems from (i) strong resistance to photobleaching allowing prolonged fluorescence observation times; (ii) the possibility to excite fluorescence using a focused electron beam (cathodoluminescence; CL) for high-resolution localization; and (iii) the potential use for nanoscale sensing. For all these schemes, the development of versatile molecular labeling using relatively small diamonds is essential. Here, we show the direct targeting of a biological molecule with nanodiamonds as small as 70 nm using a streptavidin conjugation and standard antibody labelling approach. We also show internalization of 40 nm sized nanodiamonds. The fluorescence from the nanodiamonds survives osmium-fixation and plastic embedding making them suited for correlative light and electron microscopy. We show that CL can be observed from epon-embedded nanodiamonds, while surface-exposed nanoparticles also stand out in secondary electron (SE) signal due to the exceptionally high diamond SE yield. Finally, we demonstrate the magnetic read-out using fluorescence from diamonds prior to embedding. Thus, our results firmly establish nanodiamonds containing nitrogen-vacancy centers as unique, versatile probes for combining and correlating different types of microscopy, from fluorescence imaging and magnetometry to ultrastructural investigation using electron microscopy.

Atomic resolution three-dimensional electron diffraction microscopy

International Nuclear Information System (INIS)

Miao Jianwei; Ohsuna, Tetsu; Terasaki, Osamu; Hodgson, Keith O.; O'Keefe, Michael A.

2002-01-01

We report the development of a novel form of diffraction-based 3D microscopy to overcome resolution barriers inherent in high-resolution electron microscopy and tomography. By combining coherent electron diffraction with the oversampling phasing method, we show that the 3D structure of a nanocrystal can be determined ab initio at a resolution of 1 Angstrom from 29 simulated noisy diffraction patterns. This new form of microscopy can be used to image the 3D structures of nanocrystals and noncrystalline samples, with resolution limited only by the quality of sample diffraction

Head-facial hemangiomas studied with scanning electron microscopy.

Science.gov (United States)

Cavallotti, Carlo; Cavallotti, Chiara; Giovannetti, Filippo; Iannetti, Giorgio

2009-11-01

Hemangiomas of the head or face are a frequent vascular pathology, consisting in an embryonic dysplasia that involves the cranial-facial vascular network. Hemangiomas show clinical, morphological, developmental, and structural changes during their course. Morphological, structural, ultrastructural, and clinical characteristics of head-facial hemangiomas were studied in 28 patients admitted in our hospital. Nineteen of these patients underwent surgery for the removal of the hemangiomas, whereas 9 patients were not operated on. All the removed tissues were transferred in our laboratories for the morphological staining. Light microscopy, transmission electron microscopy, and scanning electron microscopy techniques were used for the observation of all microanatomical details. All patients were studied for a clinical diagnosis, and many were subjected to surgical therapy. The morphological results revealed numerous microanatomical characteristics of the hemangiomatous vessels. The observation by light microscopy shows the afferent and the efferent vessels for every microhemangioma. All the layers of the arterial wall are uneven. The lumen of the arteriole is entirely used by a blood clot. The observation by transmission electron microscopy shows that it was impossible to see the limits of the different layers (endothelium, medial layer, and adventitia) in the whole wall of the vessels. Moreover, both the muscular and elastic components are disarranged and replaced with connective tissue. The observation by scanning electron microscopy shows that the corrosion cast of the hemangioma offers 3 periods of filling: initially with partial filling of the arteriolar and of the whole cast, intermediate with the entire filling of the whole cast (including arteriole and venule), and a last period with a partial emptying of the arteriolar and whole cast while the venule remains totally injected with resin. Our morphological results can be useful to clinicians for a precise

Immunomicroscopic localization of aminopeptidase N in the pig enterocyte. Implications for the route of intracellular transport

DEFF Research Database (Denmark)

Hansen, Gert Helge; Sjöström, H; Norén, Ove

1987-01-01

The subcellular localization of aminopeptidase N (EC 3.4.11.2) in the pig enterocyte was investigated by immunofluorescence and immunoelectron microscopy (immunogold staining). By indirect immunofluorescence on either frozen or paraffin-embedded sections, a very intense staining in the microvillar....... Labelling was demonstrated in the Golgi apparatus and in a minor fraction of the intracellular smooth vesicles positioned between the Golgi apparatus and the microvillar membrane. These observations are compatible with the view that newly synthesized aminopeptidase N is delivered directly to the microvillar...

Histophilus somni biofilm formation in cardiopulmonary tissue of the bovine host following respiratory challenge

DEFF Research Database (Denmark)

Sandal, Indra; Shao, Jian Q.; Annadata, Satish

2009-01-01

Biofilms form in a variety of host sites following infection with many bacterial species. However, the study of biofilms in a host is hindered due to the lack of protocols for the proper experimental investigation of biofilms in vivo. Histophilus somni is an agent of respiratory and systemic...... diseases in bovines, and readily forms biofilms in vitro. In the present study the capability of H. somni to form biofilms in cardiopulmonary tissue following experimental respiratory infection in the bovine host was examined by light microscopy, transmission electron microscopy, immunoelectron microscopy...... haemagglutinin (FHA), predicted to be involved in attachment. Thus, this investigation demonstrated that H. somni is capable of forming a biofilm in its natural host, that such a biofilm may be capable of harboring other bovine respiratory disease pathogens, and that the genes responsible for biofilm formation...

Transmission electron microscope studies of the nuclear envelope in Caenorhabditis elegans embryos.

Science.gov (United States)

Cohen, Merav; Tzur, Yonatan B; Neufeld, Esther; Feinstein, Naomi; Delannoy, Michael R; Wilson, Katherine L; Gruenbaum, Yosef

2002-01-01

Nuclear membranes and nuclear pore complexes (NPCs) are conserved in both animals and plants. However, the lamina composition and the dimensions of NPCs vary between plants, yeast, and vertebrates. In this study, we established a protocol that preserves the structure of Caenorhabditis elegans embryonic cells for high-resolution studies with thin-section transmission electron microscopy (TEM). We show that the NPCs are bigger in C. elegans embryos than in yeast, with dimensions similar to those in higher eukaryotes. We also localized the C. elegans nuclear envelope proteins Ce-lamin and Ce-emerin by pre-embedding gold labeling immunoelectron microscopy. Both proteins are present at or near the inner nuclear membrane. A fraction of Ce-lamin, but not Ce-emerin, is present in the nuclear interior. Removing the nuclear membranes leaves both Ce-lamin and Ce-emerin associated with the chromatin. Eliminating the single lamin protein caused cell death as visualized by characteristic changes in nuclear architecture including condensation of chromatin, clustering of NPCs, membrane blebbing, and the presence of vesicles inside the nucleus. Taken together, these results show evolutionarily conserved protein localization, interactions, and functions of the C. elegans nuclear envelope.

Simultaneous ultrastructural analysis of fluorochrome-photoconverted diaminobenzidine and gold immunolabeling in cultured cells

Directory of Open Access Journals (Sweden)

M. Malatesta

2013-09-01

Full Text Available Diaminobenzidine photoconversion is a technique by which a fluorescent dye is transformed into a stably insoluble, brown, electrondense signal, thus enabling examination at both bright field light microscopy and transmission electron microscopy. In this work, a procedure is proposed for combining photoconversion and immunoelectron microscopy: in vitro cell cultures have been first submitted to photoconversion to analyse the intracellular fate of either fluorescent nanoparticles or photosensitizing molecules, then processed for transmission electron microscopy; different fixative solutions and embedding media have been used, and the ultrathin sections were finally submitted to post-embedding immunogold cytochemistry. Under all conditions the photoconversion reaction product and the target antigen were properly detected in the same section; Epon-embedded, osmicated samples required a pre-treatment with sodium metaperiodate to unmask the antigenic sites. This simple and reliable procedure exploits a single sample to simultaneously localise the photoconversion product and a variety of antigens allowing a specific identification of subcellular organelles at the ultrastructural level.

Similar chemokine receptor profiles in lymphomas with central nervous system involvement - possible biomarkers for patient selection for central nervous system prophylaxis, a retrospective study.

Science.gov (United States)

Lemma, Siria A; Pasanen, Anna Kaisa; Haapasaari, Kirsi-Maria; Sippola, Antti; Sormunen, Raija; Soini, Ylermi; Jantunen, Esa; Koivunen, Petri; Salokorpi, Niina; Bloigu, Risto; Turpeenniemi-Hujanen, Taina; Kuittinen, Outi

2016-05-01

Central nervous system (CNS) relapse occurs in around 5% of diffuse large B-cell lymphoma (DLBCL) cases. No biomarkers to identify high-risk patients have been discovered. We evaluated the expression of lymphocyte-guiding chemokine receptors in systemic and CNS lymphomas. Immunohistochemical staining for CXCR4, CXCR5, CCR7, CXCL12, and CXCL13 was performed on 89 tissue samples, including cases of primary central nervous system lymphoma (PCNSL), secondary CNS lymphoma (sCNSL), and systemic DLBCL. Also, 10 reactive lymph node samples were included. Immunoelectron microscopy was performed on two PCNSLs, one sCNSL, one systemic DLBCL, and one reactive lymph node samples, and staining was performed for CXCR4, CXCR5, CXCL12, and CXCL13. Chi-square test was used to determine correlations between clinical parameters, diagnostic groups, and chemokine receptor expression. Strong nuclear CXCR4 positivity correlated with systemic DLBCL, whereas strong cytoplasmic CXCR5 positivity correlated with CNS involvement (P = 0.003 and P = 0.039). Immunoelectron microscopy revealed a nuclear CXCR4 staining in reactive lymph node, compared with cytoplasmic and membranous localization seen in CNS lymphomas. We found that CNS lymphoma presented a chemokine receptor profile different from systemic disease. Our findings give new information on the CNS tropism of DLBCL and, if confirmed, may contribute to more effective targeting of CNS prophylaxis among patients with DLBCL. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Spectroscopy and atomic force microscopy of biomass.

Science.gov (United States)

Tetard, L; Passian, A; Farahi, R H; Kalluri, U C; Davison, B H; Thundat, T

2010-05-01

Scanning probe microscopy has emerged as a powerful approach to a broader understanding of the molecular architecture of cell walls, which may shed light on the challenge of efficient cellulosic ethanol production. We have obtained preliminary images of both Populus and switchgrass samples using atomic force microscopy (AFM). The results show distinctive features that are shared by switchgrass and Populus. These features may be attributable to the lignocellulosic cell wall composition, as the collected images exhibit the characteristic macromolecular globule structures attributable to the lignocellulosic systems. Using both AFM and a single case of mode synthesizing atomic force microscopy (MSAFM) to characterize Populus, we obtained images that clearly show the cell wall structure. The results are of importance in providing a better understanding of the characteristic features of both mature cells as well as developing plant cells. In addition, we present spectroscopic investigation of the same samples.

Progress in the Correlative Atomic Force Microscopy and Optical Microscopy

Directory of Open Access Journals (Sweden)

Lulu Zhou

2017-04-01

Full Text Available Atomic force microscopy (AFM has evolved from the originally morphological imaging technique to a powerful and multifunctional technique for manipulating and detecting the interactions between molecules at nanometer resolution. However, AFM cannot provide the precise information of synchronized molecular groups and has many shortcomings in the aspects of determining the mechanism of the interactions and the elaborate structure due to the limitations of the technology, itself, such as non-specificity and low imaging speed. To overcome the technical limitations, it is necessary to combine AFM with other complementary techniques, such as fluorescence microscopy. The combination of several complementary techniques in one instrument has increasingly become a vital approach to investigate the details of the interactions among molecules and molecular dynamics. In this review, we reported the principles of AFM and optical microscopy, such as confocal microscopy and single-molecule localization microscopy, and focused on the development and use of correlative AFM and optical microscopy.

Subcellular localization of Patched and Smoothened, the receptors for Sonic hedgehog signaling, in the hippocampal neuron.

Science.gov (United States)

Petralia, Ronald S; Schwartz, Catherine M; Wang, Ya-Xian; Mattson, Mark P; Yao, Pamela J

2011-12-15

Cumulative evidence suggests that, aside from patterning the embryonic neural tube, Sonic hedgehog (Shh) signaling plays important roles in the mature nervous system. In this study, we investigate the expression and localization of the Shh signaling receptors, Patched (Ptch) and Smoothened (Smo), in the hippocampal neurons of young and mature rats. Reverse transcriptase-polymerase chain reaction and immunoblotting analyses show that the expression of Ptch and Smo remains at a moderate level in young postnatal and adult brains. By using immunofluorescence light microscopy and immunoelectron microscopy, we examine the spatial distribution of Ptch and Smo within the hippocampal neurons. In young developing neurons, Ptch and Smo are present in the processes and are clustered at their growth cones. In mature neurons, Ptch and Smo are concentrated in dendrites, spines, and postsynaptic sites. Synaptic Ptch and Smo often co-exist with unusual structures-synaptic spinules and autophagosomes. Our results reveal the anatomical organization of the Shh receptors within both the young and the mature hippocampal neurons. Copyright © 2011 Wiley-Liss, Inc.

Subcellular localization of YKL-40 in normal and malignant epithelial cells of the breast

DEFF Research Database (Denmark)

Roslind, A.; Balslev, E.; Kruse, H.

2008-01-01

. YKL-40 protein expression was redistributed in carcinoma versus normal glandular tissue of the breast. A reduced expression of YKL-40 in relation to intermediate filaments and desmosomes was found in tumor cells. Changes in YKL-40 expression suggest that the function of YKL-40 in cells of epithelial......YKL-40 is a new prognostic biomarker in cancer. The biological function is only poorly understood. This study aimed at determining the subcellular localization of YKL-40, using immunogold labeling, in normal epithelial cells and in malignant tumor cells of the breast by immunoelectron microscopy...

Molecular biology of the Chlamydia pneumoniae surface

DEFF Research Database (Denmark)

Christiansen, Gunna; Østergaard, Lars; Birkelund, Svend

1997-01-01

surface differs from that of Chlamydia trachomatis. In order to study the surface of C. pneumoniae we generated monoclonal antibodies (MAbs) against C. pneumoniae strain VR-1310 and selected 14 MAbs that reacted with the surface of C. pneumoniae. All MAbs reacted in immunoelectron microscopy...... with the surface of both whole C. pneumoniae VR-1310 elementary bodies and with purified sarcosyl extracted outer membrane complexes. However, only 2 of the MAbs reacted in immunoblotting with C. pneumoniae proteins and only with antigen that had not been heat treated in SDS-sample buffer. This indicates...

Demonstration of an orexinergic central innervation of the pineal gland of the pig

DEFF Research Database (Denmark)

Fabris, Chiara; Cozzi, Bruno; Hay-Schmidt, Anders

2004-01-01

into the pineal stalk and parenchyma to disperse among the pinealocytes. Immunoelectron microscopy confirmed the presence of orexinergic nerve fibers in the pig pineal gland. After extraction of total mRNA from the hypothalamus and pineal gland, we performed RT-PCR and nested PCR using primers specific...... for porcine orexin receptors. PCR products were sequenced, verifying the presence of both OR-R1 and OR-R2 in the tissues investigated. These findings, supported by previous studies on rodents, suggest a hypothalamic regulation of the pineal gland via central orexinergic nervous inputs....

High resolution scanning electron microscopy of rabbit corneal endothelium to show effects of UV-visible irradiation in the presence of chlorpromazine

Energy Technology Data Exchange (ETDEWEB)

Lea, P.J.; Hollenberg, M.J.; Menon, I.A.; Temkin, R.J.; Persad, S.D.; Basu, P.K. (Univ. of Toronto, Ontario (Canada))

1989-01-01

The ultrastructure of rabbit cornea endothelial cells was examined by scanning electron microscopy (SEM) in freeze-cleaved corneas using a Hitachi S-570 scanning electron microscope in the high resolution mode (HRSEM). In order to study phototoxic effects in vitro, rabbit corneas (experimental) were cultured as organ culture in the presence of 5 micrograms/ml chlorpromazine (CPZ) and irradiated. For comparison, control 1 corneas were not irradiated but incubated in the dark without CPZ in the medium; control 2 corneas were also kept in the dark but in the presence of CPZ; control 3 corneas were irradiated with no CPZ in the medium. Cellular damage was not seen in the three types of control corneas, but in the experimental corneas the endothelial cells showed extensive disruption of the cell membrane and some deterioration of the intracellular components. Our study confirmed that HRSEM is a satisfactory new technique for visualizing damage of the intracellular organelles of corneal endothelium.

High resolution scanning electron microscopy of rabbit corneal endothelium to show effects of UV-visible irradiation in the presence of chlorpromazine

International Nuclear Information System (INIS)

Lea, P.J.; Hollenberg, M.J.; Menon, I.A.; Temkin, R.J.; Persad, S.D.; Basu, P.K.

1989-01-01

The ultrastructure of rabbit cornea endothelial cells was examined by scanning electron microscopy (SEM) in freeze-cleaved corneas using a Hitachi S-570 scanning electron microscope in the high resolution mode (HRSEM). In order to study phototoxic effects in vitro, rabbit corneas (experimental) were cultured as organ culture in the presence of 5 micrograms/ml chlorpromazine (CPZ) and irradiated. For comparison, control 1 corneas were not irradiated but incubated in the dark without CPZ in the medium; control 2 corneas were also kept in the dark but in the presence of CPZ; control 3 corneas were irradiated with no CPZ in the medium. Cellular damage was not seen in the three types of control corneas, but in the experimental corneas the endothelial cells showed extensive disruption of the cell membrane and some deterioration of the intracellular components. Our study confirmed that HRSEM is a satisfactory new technique for visualizing damage of the intracellular organelles of corneal endothelium

CARS microscopy of Alzheimer's diseased brain tissue

Science.gov (United States)

Enejder, Annika; Kiskis, Juris; Fink, Helen; Nyberg, Lena; Thyr, Jakob; Li, Jia-Yi

2014-02-01

Alzheimer's disease (AD) is a progressive neurodegenerative disorder currently without cure, characterized by the presence of extracellular plaques surrounded by dystrophic neurites. In an effort to understand the underlying mechanisms, biochemical analysis (protein immunoblot) of plaque extracts reveals that they consist of amyloid-beta (Aβ) peptides assembled as oligomers, protofibrils and aggregates. Their spatial distribution has been confirmed by Thioflavin-S or immuno-staining with fluorescence microscopy. However, it is increasingly understood that the protein aggregation is only one of several mechanism that causes neuronal dysfunction and death. This raises the need for a more complete biochemical analysis. In this study, we have complemented 2-photon fluorescence microscopy of Thioflavin-S and Aβ immuno-stained human AD plaques with CARS microscopy. We show that the chemical build-up of AD plaques is more complex and that Aβ staining does not provide the complete picture of the spatial distribution or the molecular composition of AD plaques. CARS images provide important complementary information to that obtained by fluorescence microscopy, motivating a broader introduction of CARS microscopy in the AD research field.

French Society of Microscopy, 10. conference; Societe Francaise des Microscopies, 10. colloque

Energy Technology Data Exchange (ETDEWEB)

Thibault-Penisson, J; Cremer, Ch; Susini, J; Kirklanda, A I; Rigneault, H; Renault, O; Bailly, A; Zagonel, L F; Barrett, N; Bogner, A; Gauthier, C; Jouneau, P H; Thollet, G; Fuchs, G; Basset, D; Deconihout, B; Vurpillot, F; Vella, A; Matthieu, G; Cadel, E; Bostel, A; Blavette, D; Baumeister, W; Usson, Y; Zaefferer, St; Laffont, L; Weyland, M; Thomas, J M; Midgley, P; Benlekbir, S; Epicier, Th; Diop, B N; Roux, St; Ou, M; Perriat, P; Bausach, M; Aouine, M; Berhault, G; Idrissi, H; Cottevieille, M; Jonic, S; Larquet, E; Svergun, D; Vannoni, M A; Boisset, N; Ersena, O; Werckmann, J; Ulhaq, C; Hirlimann, Ch; Tihay, F; Cuong, Pham-Huu; Crucifix, C; Schultz, P; Jornsanoha, P; Thollet, G; Masenelli-Varlot, K; Gauthier, C; Ludwig, W; King, A; Johnson, G; Gonzalves-Hoennicke, M; Reischig, P; Messaoudi, C; Ibrahim, R; Marco, S; Klie, R F; Zhao, Y; Yang, G; Zhu, Y; Hue, F; Hytch, M; Hartmann, J M; Bogumilowicz, Y; Claverie, A; Klein, H; Alloyeau, D; Ricolleau, C; Langlois, C; Le Bouar, Y; Loiseau, A; Colliex, C; Stephan, O; Kociak, M; Tence, M; Gloter, A; Imhoff, D; Walls, M; Nelayah, J; March, K; Couillard, M; Ailliot, C; Bertin, F; Cooper, D; Rivallin, P; Dumelie, N; Benhayoune, H; Balossier, G; Cheynet, M; Pokrant, S; Tichelaar, F; Rouviere, J L; Cooper, D; Truche, R; Chabli, A; Debili, M Y; Houdellier, F; Warot-Fonrose, B; Hytch, M J; Snoeck, E; Calmels, L; Serin, V; Schattschneider, P; Jacob, D; Cordier, P

2007-07-01

This document gathers the resumes of some of the presentations made at this conference whose aim was to present the last developments and achievements of the 3 complementary microscopies: optical microscopy, electron microscopy and X-ray microscopy. The contributions have been organized around the following 12 topics: 1) new technical developments, 2) 3-dimensional imaging, 3) quantitative microscopy, 4) technical progress in photon microscopy, 5) synchrotron radiation, 6) measurements of patterns, deformations and strains, 7) materials for energy and transports, 8) nano-structures, 9) virus: structure and infection mechanisms, 10) 3-dimensional imaging for molecules, cells and cellular tissues, 11) nano-particles and colloids, and 12) liquid crystals.

Association of TCTP with Centrosome and Microtubules

Directory of Open Access Journals (Sweden)

Mariusz K. Jaglarz

2012-01-01

Full Text Available Translationally Controlled Tumour Protein (TCTP associates with microtubules (MT, however, the details of this association are unknown. Here we analyze the relationship of TCTP with MTs and centrosomes in Xenopus laevis and mammalian cells using immunofluorescence, tagged TCTP expression and immunoelectron microscopy. We show that TCTP associates both with MTs and centrosomes at spindle poles when detected by species-specific antibodies and by Myc-XlTCTP expression in Xenopus and mammalian cells. However, when the antibodies against XlTCTP were used in mammalian cells, TCTP was detected exclusively in the centrosomes. These results suggest that a distinct pool of TCTP may be specific for, and associate with, the centrosomes. Double labelling for TCTP and γ-tubulin with immuno-gold electron microscopy in Xenopus laevis oogonia shows localization of TCTP at the periphery of the γ-tubulin-containing pericentriolar material (PCM enveloping the centriole. TCTP localizes in the close vicinity of, but not directly on the MTs in Xenopus ovary suggesting that this association requires unidentified linker proteins. Thus, we show for the first time: (1 the association of TCTP with centrosomes, (2 peripheral localization of TCTP in relation to the centriole and the γ-tubulin-containing PCM within the centrosome, and (3 the indirect association of TCTP with MTs.

Handheld Fluorescence Microscopy based Flow Analyzer.

Science.gov (United States)

Saxena, Manish; Jayakumar, Nitin; Gorthi, Sai Siva

2016-03-01

Fluorescence microscopy has the intrinsic advantages of favourable contrast characteristics and high degree of specificity. Consequently, it has been a mainstay in modern biological inquiry and clinical diagnostics. Despite its reliable nature, fluorescence based clinical microscopy and diagnostics is a manual, labour intensive and time consuming procedure. The article outlines a cost-effective, high throughput alternative to conventional fluorescence imaging techniques. With system level integration of custom-designed microfluidics and optics, we demonstrate fluorescence microscopy based imaging flow analyzer. Using this system we have imaged more than 2900 FITC labeled fluorescent beads per minute. This demonstrates high-throughput characteristics of our flow analyzer in comparison to conventional fluorescence microscopy. The issue of motion blur at high flow rates limits the achievable throughput in image based flow analyzers. Here we address the issue by computationally deblurring the images and show that this restores the morphological features otherwise affected by motion blur. By further optimizing concentration of the sample solution and flow speeds, along with imaging multiple channels simultaneously, the system is capable of providing throughput of about 480 beads per second.

Fluctuation microscopy analysis of amorphous silicon models

Energy Technology Data Exchange (ETDEWEB)

Gibson, J.M., E-mail: jmgibson@fsu.edu [Northeastern University, Department of Physics, Boston MA 02115 (United States); FAMU/FSU Joint College of Engineering, 225 Pottsdamer Street, Tallahassee, FL 32310 (United States); Treacy, M.M.J. [Arizona State University, Department of Physics, Tempe AZ 85287 (United States)

2017-05-15

Highlights: • Studied competing computer models for amorphous silicon and simulated fluctuation microscopy data. • Show that only paracrystalline/random network composite can fit published data. • Specifically show that pure random network or random network with void models do not fit available data. • Identify a new means to measure volume fraction of ordered material. • Identify unreported limitations of the Debye model for simulating fluctuation microscopy data. - Abstract: Using computer-generated models we discuss the use of fluctuation electron microscopy (FEM) to identify the structure of amorphous silicon. We show that a combination of variable resolution FEM to measure the correlation length, with correlograph analysis to obtain the structural motif, can pin down structural correlations. We introduce the method of correlograph variance as a promising means of independently measuring the volume fraction of a paracrystalline composite. From comparisons with published data, we affirm that only a composite material of paracrystalline and continuous random network that is substantially paracrystalline could explain the existing experimental data, and point the way to more precise measurements on amorphous semiconductors. The results are of general interest for other classes of disordered materials.

Fluctuation microscopy analysis of amorphous silicon models

International Nuclear Information System (INIS)

Gibson, J.M.; Treacy, M.M.J.

2017-01-01

Highlights: • Studied competing computer models for amorphous silicon and simulated fluctuation microscopy data. • Show that only paracrystalline/random network composite can fit published data. • Specifically show that pure random network or random network with void models do not fit available data. • Identify a new means to measure volume fraction of ordered material. • Identify unreported limitations of the Debye model for simulating fluctuation microscopy data. - Abstract: Using computer-generated models we discuss the use of fluctuation electron microscopy (FEM) to identify the structure of amorphous silicon. We show that a combination of variable resolution FEM to measure the correlation length, with correlograph analysis to obtain the structural motif, can pin down structural correlations. We introduce the method of correlograph variance as a promising means of independently measuring the volume fraction of a paracrystalline composite. From comparisons with published data, we affirm that only a composite material of paracrystalline and continuous random network that is substantially paracrystalline could explain the existing experimental data, and point the way to more precise measurements on amorphous semiconductors. The results are of general interest for other classes of disordered materials.

Cryo-Scanning Electron Microscopy (SEM) and Scanning Transmission Electron Microscopy (STEM)-in-SEM for Bio- and Organo-Mineral Interface Characterization in the Environment.

Science.gov (United States)

Wille, Guillaume; Hellal, Jennifer; Ollivier, Patrick; Richard, Annie; Burel, Agnes; Jolly, Louis; Crampon, Marc; Michel, Caroline

2017-12-01

Understanding biofilm interactions with surrounding substratum and pollutants/particles can benefit from the application of existing microscopy tools. Using the example of biofilm interactions with zero-valent iron nanoparticles (nZVI), this study aims to apply various approaches in biofilm preparation and labeling for fluorescent or electron microscopy and energy dispersive X-ray spectrometry (EDS) microanalysis for accurate observations. According to the targeted microscopy method, biofilms were sampled as flocs or attached biofilm, submitted to labeling using 4',6-diamidino-2-phenylindol, lectins PNA and ConA coupled to fluorescent dye or gold nanoparticles, and prepared for observation (fixation, cross-section, freezing, ultramicrotomy). Fluorescent microscopy revealed that nZVI were embedded in the biofilm structure as aggregates but the resolution was insufficient to observe individual nZVI. Cryo-scanning electron microscopy (SEM) observations showed nZVI aggregates close to bacteria, but it was not possible to confirm direct interactions between nZVI and cell membranes. Scanning transmission electron microscopy in the SEM (STEM-in-SEM) showed that nZVI aggregates could enter the biofilm to a depth of 7-11 µm. Bacteria were surrounded by a ring of extracellular polymeric substances (EPS) preventing direct nZVI/membrane interactions. STEM/EDS mapping revealed a co-localization of nZVI aggregates with lectins suggesting a potential role of EPS in nZVI embedding. Thus, the combination of divergent microscopy approaches is a good approach to better understand and characterize biofilm/metal interactions.

Observation of self-assembled fluorescent beads by scanning near-field optical microscopy and atomic force microscopy

International Nuclear Information System (INIS)

Oh, Y.J.; Jo, W.; Kim, Min-Gon; Kyu Park, Hyun; Hyun Chung, Bong

2006-01-01

Optical response and topography of fluorescent latex beads both on flat self-assembled monolayer and on a micron-patterned surface with poly(dimethylsiloxane) are studied. Scanning near-field optical microscopy and atomic force microscopy were utilized together for detecting fluorescence and imaging topography of the patterned latex beads, respectively. As a result, the micro-patterned latex beads where a specific chemical binding occurred show a strong signal, whereas no signals are observed in the case of nonspecific binding. With fluorescein isothiocyanate (FITC), it is convenient to measure fluorescence signal from the patterned beads allowing us to monitor the small balls of fluorescent latex

Coherent light microscopy

CERN Document Server

Ferraro, Pietro; Zalevsky, Zeev

2011-01-01

This book deals with the latest achievements in the field of optical coherent microscopy. While many other books exist on microscopy and imaging, this book provides a unique resource dedicated solely to this subject. Similarly, many books describe applications of holography, interferometry and speckle to metrology but do not focus on their use for microscopy. The coherent light microscopy reference provided here does not focus on the experimental mechanics of such techniques but instead is meant to provide a users manual to illustrate the strengths and capabilities of developing techniques. Th

Correlating Intravital Multi-Photon Microscopy to 3D Electron Microscopy of Invading Tumor Cells Using Anatomical Reference Points

Science.gov (United States)

Karreman, Matthia A.; Mercier, Luc; Schieber, Nicole L.; Shibue, Tsukasa; Schwab, Yannick; Goetz, Jacky G.

2014-01-01

Correlative microscopy combines the advantages of both light and electron microscopy to enable imaging of rare and transient events at high resolution. Performing correlative microscopy in complex and bulky samples such as an entire living organism is a time-consuming and error-prone task. Here, we investigate correlative methods that rely on the use of artificial and endogenous structural features of the sample as reference points for correlating intravital fluorescence microscopy and electron microscopy. To investigate tumor cell behavior in vivo with ultrastructural accuracy, a reliable approach is needed to retrieve single tumor cells imaged deep within the tissue. For this purpose, fluorescently labeled tumor cells were subcutaneously injected into a mouse ear and imaged using two-photon-excitation microscopy. Using near-infrared branding, the position of the imaged area within the sample was labeled at the skin level, allowing for its precise recollection. Following sample preparation for electron microscopy, concerted usage of the artificial branding and anatomical landmarks enables targeting and approaching the cells of interest while serial sectioning through the specimen. We describe here three procedures showing how three-dimensional (3D) mapping of structural features in the tissue can be exploited to accurately correlate between the two imaging modalities, without having to rely on the use of artificially introduced markers of the region of interest. The methods employed here facilitate the link between intravital and nanoscale imaging of invasive tumor cells, enabling correlating function to structure in the study of tumor invasion and metastasis. PMID:25479106

Shedding light on endocytosis with optimized super-resolution microscopy

NARCIS (Netherlands)

Leyton Puig, D.M.

2017-01-01

Super-resolution microscopy is a relatively new microscopy technique that is still under optimization. In this thesis we focus on the improvement of the quality of super-resolution images, to apply them to the study of the processes of cell signaling and endocytosis. First, we show that the use of a

Basement membrane-specific chondroitin sulfate proteoglycan is abnormally associated with the glomerular capillary basement membrane of diabetic rats

DEFF Research Database (Denmark)

McCarthy, K J; Abrahamson, D R; Bynum, K R

1994-01-01

exception being the normal glomerular capillary basement membrane (GBM), where it is absent. In the present study of mature kidneys we examined the distribution of BM-CSPG in streptozocin-induced diabetes mellitus in rats. We found BM-CSPG atypically associated with the GBM of diabetic animals as early as 1...... month after induction of diabetes mellitus. Immunoelectron microscopy (IEM) of affected capillary loops showed BM-CSPG present in the subendothelial matrix in areas of GBM thickening and absent in areas where the GBM appears to be of normal thickness. Moreover, the association of BM-CSPG with regions...... of the pericapillary GBM affects the morphology of the capillary endothelial cells within these areas, directly displacing the cell body from the GBM proper and causing loss of fenestrae. These new data on BM-CSPG distribution reflect abnormal glomerular extracellular matrix protein biosynthesis/turnover in diabetes...

Super-resolution fluorescence microscopy by stepwise optical saturation

Science.gov (United States)

Zhang, Yide; Nallathamby, Prakash D.; Vigil, Genevieve D.; Khan, Aamir A.; Mason, Devon E.; Boerckel, Joel D.; Roeder, Ryan K.; Howard, Scott S.

2018-01-01

Super-resolution fluorescence microscopy is an important tool in biomedical research for its ability to discern features smaller than the diffraction limit. However, due to its difficult implementation and high cost, the super-resolution microscopy is not feasible in many applications. In this paper, we propose and demonstrate a saturation-based super-resolution fluorescence microscopy technique that can be easily implemented and requires neither additional hardware nor complex post-processing. The method is based on the principle of stepwise optical saturation (SOS), where M steps of raw fluorescence images are linearly combined to generate an image with a M-fold increase in resolution compared with conventional diffraction-limited images. For example, linearly combining (scaling and subtracting) two images obtained at regular powers extends the resolution by a factor of 1.4 beyond the diffraction limit. The resolution improvement in SOS microscopy is theoretically infinite but practically is limited by the signal-to-noise ratio. We perform simulations and experimentally demonstrate super-resolution microscopy with both one-photon (confocal) and multiphoton excitation fluorescence. We show that with the multiphoton modality, the SOS microscopy can provide super-resolution imaging deep in scattering samples. PMID:29675306

Combined frequency modulated atomic force microscopy and scanning tunneling microscopy detection for multi-tip scanning probe microscopy applications

International Nuclear Information System (INIS)

Morawski, Ireneusz; Spiegelberg, Richard; Korte, Stefan; Voigtländer, Bert

2015-01-01

A method which allows scanning tunneling microscopy (STM) tip biasing independent of the sample bias during frequency modulated atomic force microscopy (AFM) operation is presented. The AFM sensor is supplied by an electronic circuit combining both a frequency shift signal and a tunneling current signal by means of an inductive coupling. This solution enables a control of the tip potential independent of the sample potential. Individual tip biasing is specifically important in order to implement multi-tip STM/AFM applications. An extensional quartz sensor (needle sensor) with a conductive tip is applied to record simultaneously topography and conductivity of the sample. The high resonance frequency of the needle sensor (1 MHz) allows scanning of a large area of the surface being investigated in a reasonably short time. A recipe for the amplitude calibration which is based only on the frequency shift signal and does not require the tip being in contact is presented. Additionally, we show spectral measurements of the mechanical vibration noise of the scanning system used in the investigations

Combined frequency modulated atomic force microscopy and scanning tunneling microscopy detection for multi-tip scanning probe microscopy applications

Energy Technology Data Exchange (ETDEWEB)

Morawski, Ireneusz [Peter Grünberg Institut (PGI-3) and JARA-Fundamentals of Future Information Technology, Forschungszentrum Jülich, 52425 Jülich (Germany); Institute of Experimental Physics, University of Wrocław, pl. M. Borna 9, 50-204 Wrocław (Poland); Spiegelberg, Richard; Korte, Stefan; Voigtländer, Bert [Peter Grünberg Institut (PGI-3) and JARA-Fundamentals of Future Information Technology, Forschungszentrum Jülich, 52425 Jülich (Germany)

2015-12-15

A method which allows scanning tunneling microscopy (STM) tip biasing independent of the sample bias during frequency modulated atomic force microscopy (AFM) operation is presented. The AFM sensor is supplied by an electronic circuit combining both a frequency shift signal and a tunneling current signal by means of an inductive coupling. This solution enables a control of the tip potential independent of the sample potential. Individual tip biasing is specifically important in order to implement multi-tip STM/AFM applications. An extensional quartz sensor (needle sensor) with a conductive tip is applied to record simultaneously topography and conductivity of the sample. The high resonance frequency of the needle sensor (1 MHz) allows scanning of a large area of the surface being investigated in a reasonably short time. A recipe for the amplitude calibration which is based only on the frequency shift signal and does not require the tip being in contact is presented. Additionally, we show spectral measurements of the mechanical vibration noise of the scanning system used in the investigations.

Statistical validation of individual fibre segmentation from tomograms and microscopy

DEFF Research Database (Denmark)

Emerson, Monica Jane; Dahl, Vedrana Andersen; Conradsen, Knut

2018-01-01

at different resolutions to optical microscopy (OM) and scanning electron microscopy (SEM), where we characterise fibres by their diameters and positions. In addition to comparing individual fibre diameters, we also model their spatial distribution, and compare the obtained model parameters. Our study shows...

Bauxite and bauxite residue, characterization and electron microscopy study

International Nuclear Information System (INIS)

Antunes, M.L.P.; Conceicao, F.T.; Toledo, S.P.; Kiyohara, P.K.

2012-01-01

Through the Bayer process, bauxite is refined and alumina is produced. In this process, a highly alkaline residue, red mud is generated and its disposal represents an environmental problem. The aim of this paper is to present the characterization of Brazilian bauxite and Brazilian red mud by: X-ray diffraction, specific surface area, chemical composition analysis by ICP-MS, transmission electron microscopy (TEM) and energy dispersive X-ray spectrometry (EDS), and scanning electron microscopy (SEM) and discuss possible applications of this residue. The results identify as a constituent of both materials: Al 2 O 3 , Fe 2 O 3 , TiO 2 and SiO 2 and the presence of Na 2 O in residue. The analysis by electron microscopy of Bauxite shows particles with hexagonal shape and red mud shows small particles size. (author)

Increased AAA-TOB3 correlates with lymph node metastasis and advanced stage of lung adenocarcinoma.

Science.gov (United States)

Liu, Yanfeng; Bu, Lina; Li, Wei; Wu, Wei; Wang, Shengyu; Diao, Xin; Zhou, Jing; Chen, Guoan; Yang, Shuanying

2017-07-24

This study was to investigate the differential mitochondrial protein expressions in human lung adenocarcinoma and provide preliminary data for further exploration of the carcinogenic mechanism. Total proteins of A549 and 16HBE mitochondria were extracted through 2D polyacrylamide gel electrophoresis (2-DE). The differential mitochondria proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and were further confirmed by Western blot, immunoelectron microscopy and immunohistochemistry (IHC) in A549 cells as well as lung adenocarcinoma tissues. A total of 41 differentially expressed protein spots were found in A549 mitochondria. Of them, 15 proteins were highly expressed and 26 proteins were lowly expressed in the mitochondria of A549 (by more than 1.5 times). Among the 15 more highly expressed proteins, AAA-TOB3 (by more than 3 times) was highly expressed in the mitochondria of A549 compared with the 16HBE, by LC-MS/MS identification. High electron density and clear circular colloidal gold-marked AAA-TOB3 particles were observed in the A549 cells via immunoelectron microscopy. Besides, AAA-TOB3 was confirmed to be elevated in lung adenocarcinoma by Western blot and IHC. Moreover, increased AAA-TOB3 correlated with lymph node metastasis and advanced stage of lung adenocarcinoma (pAAA-TOB3 was highly expressed in lung adenocarcinoma, and the up-regulation of AAA-TOB3 correlated with lymph node metastasis and advanced stage of lung adenocarcinoma, which suggested that it could serve as a potential molecular marker for lung adenocarcinoma.

New microscopy for nanoimaging

CERN Document Server

Kinjo, Y; Watanabe, M

2002-01-01

Two types of new microscopy, namely, X-ray contact microscopy (XRCM) in combination with atomic force microscopy (AFM) and X-ray projection microscopy (XRPM) using synchrotron radiation and zone plate optics were used to image the fine structures of human chromosomes. In the XRCM plus AFM system, location of X-ray images on a photoresist has become far easier than that with our previous method using transmission electron microscopy coupled with the replica method. In addition, the images obtained suggested that the conformation of chromatin fiber differs from the current textbook model regarding the architecture of a eukaryotic chromosome. X-ray images with high contrast of the specimens could be obtained with XRPM. The resolution of each microscopy was about 30 and 200-300 nm for XRCM plus AFM and XRPM, respectively. (author)

Graphene-enabled electron microscopy and correlated super-resolution microscopy of wet cells.

Science.gov (United States)

Wojcik, Michal; Hauser, Margaret; Li, Wan; Moon, Seonah; Xu, Ke

2015-06-11

The application of electron microscopy to hydrated biological samples has been limited by high-vacuum operating conditions. Traditional methods utilize harsh and laborious sample dehydration procedures, often leading to structural artefacts and creating difficulties for correlating results with high-resolution fluorescence microscopy. Here, we utilize graphene, a single-atom-thick carbon meshwork, as the thinnest possible impermeable and conductive membrane to protect animal cells from vacuum, thus enabling high-resolution electron microscopy of wet and untreated whole cells with exceptional ease. Our approach further allows for facile correlative super-resolution and electron microscopy of wet cells directly on the culturing substrate. In particular, individual cytoskeletal actin filaments are resolved in hydrated samples through electron microscopy and well correlated with super-resolution results.

Electron microscopy of surfaces

International Nuclear Information System (INIS)

Venables, J.A.

1981-01-01

Electron beam techniques used to study clean surfaces and surface processes on a microscopic scale are reviewed. Recent experimental examples and possible future developments are discussed. Special emphasis is given to (i) transmission diffraction and microscopy techniques, including atomic imaging; (ii) Auger microscopy on bulk and thin film samples; (iii) secondary electron microscopy, especially low energy secondaries for work-function imaging and photoelectron imaging; and (iv) reflection electron microscopy and diffraction. (orig.)

X-ray microscopy in Aarhus

International Nuclear Information System (INIS)

Uggerhoej, Erik; Abraham-Peskir, Joanna V.

2000-01-01

The Aarhus imaging soft X-ray microscope is now a busy multi-user facility. The optical set-up will be described and project highlights discussed. a) Metal-induced structural changes in whole cells in solution. The effects of aluminum, copper, nickel and zinc on protozoa investigated by using a combination of light microscopy, confocal scanning laser microscopy and X-ray microscopy. b) Botanical studies by X-ray microscopy used to compliment electron microscopy studies. c) Sludge morphology and iron precipitation in Danish freshwater plants by combining X-ray, scanning electron and transmission electron microscopy

Advanced Microscopy of Microbial Cells

DEFF Research Database (Denmark)

Haagensen, Janus Anders Juul; Regenberg, Birgitte; Sternberg, Claus

2011-01-01

microscopy, super-resolution optical microscopy (STED, SIM, PALM) as well as atomic force microscopy and Raman spectroscopy. Using examples of bistability in microbial populations as well as biofilm development and differentiation in bacterial and yeast consortia, we demonstrate the importance of microscopy...

Subsurface measurement of nanostructures on GaAs by electrostatic force microscopy

International Nuclear Information System (INIS)

Yamada, Fumihiko; Kamiya, Itaru

2013-01-01

The size of surface buried oxide nanostructures are measured by electrostatic force microscopy (EFM). In contrast to atomic force microscopy that cannot probe subsurface structures and thickness, we show that EFM data include information about the thickness of individual nanostructures, consequently allowing us to determine the thickness of buried nanostructures on semiconductor substrates. We further show that this measurement can be performed simultaneously with AFM using EFM modulation spectroscopy.

Photosynthetic and Heterotrophic Ferredoxin Isoproteins Are Colocalized in Fruit Plastids of Tomato1

Science.gov (United States)

Aoki, Koh; Yamamoto, Miyuki; Wada, Keishiro

1998-01-01

Fruit tissues of tomato (Lycopersicon esculentum Mill.) contain both photosynthetic and heterotrophic ferredoxin (FdA and FdE, respectively) isoproteins, irrespective of their photosynthetic competence, but we did not previously determine whether these proteins were colocalized in the same plastids. In isolated fruit chloroplasts and chromoplasts, both FdA and FdE were detected by immunoblotting. Colocalization of FdA and FdE in the same plastids was demonstrated using double-staining immunofluorescence microscopy. We also found that FdA and FdE were colocalized in fruit chloroplasts and chloroamyloplasts irrespective of sink status of the plastid. Immunoelectron microscopy demonstrated that FdA and FdE were randomly distributed within the plastid stroma. To investigate the significance of the heterotrophic Fd in fruit plastids, Glucose 6-phosphate dehydrogenase (G6PDH) activity was measured in isolated fruit and leaf plastids. Fruit chloroplasts and chromoplasts showed much higher G6PDH activity than did leaf chloroplasts, suggesting that high G6PDH activity is linked with FdE to maintain nonphotosynthetic production of reducing power. This result suggested that, despite their morphological resemblance, fruit chloroplasts are functionally different from their leaf counterparts. PMID:9765529

Confocal Raman microscopy

CERN Document Server

Dieing, Thomas; Hollricher, Olaf

2018-01-01

This second edition provides a cutting-edge overview of physical, technical and scientific aspects related to the widely used analytical method of confocal Raman microscopy. The book includes expanded background information and adds insights into how confocal Raman microscopy, especially 3D Raman imaging, can be integrated with other methods to produce a variety of correlative microscopy combinations. The benefits are then demonstrated and supported by numerous examples from the fields of materials science, 2D materials, the life sciences, pharmaceutical research and development, as well as the geosciences.

Scanning electron microscopy of coal macerals

Energy Technology Data Exchange (ETDEWEB)

Davis, M.R.; White, A.; Deegan, M.D.

1986-02-01

Individual macerals separated from some United Kingdom coals of Carboniferous age and bituminous rank were examined by scanning electron microscopy. In each case a specific morphology characteristic of the macerals studied could be recognized. Collinite (a member of the vitrinite maceral group) was recognizable in all samples by its angular shape and characteristic fracture patterns, the particles (30-200 ..mu..m) frequently showing striated or laminated surface. Sporinite particles had no well defined shape and were associated with more detrital material than were the other macerals studied. This detritus was shown by conventional light microscopy to be the maceral micrinite. Fusinite was remarkable in having a chunky needle form, with lengths of up to 200 ..mu..m. 8 references.

Advanced Microscopy of Microbial Cells

DEFF Research Database (Denmark)

Haagensen, Janus Anders Juul; Regenberg, Birgitte; Sternberg, Claus

2011-01-01

Growing awareness of heterogeneity in cells of microbial populations has emphasized the importance of advanced microscopy for visualization and understanding of the molecular mechanisms underlying cell-to-cell variation. In this review, we highlight some of the recent advances in confocal...... microscopy, super-resolution optical microscopy (STED, SIM, PALM) as well as atomic force microscopy and Raman spectroscopy. Using examples of bistability in microbial populations as well as biofilm development and differentiation in bacterial and yeast consortia, we demonstrate the importance of microscopy...

Saturated virtual fluorescence emission difference microscopy based on detector array

Science.gov (United States)

Liu, Shaocong; Sun, Shiyi; Kuang, Cuifang; Ge, Baoliang; Wang, Wensheng; Liu, Xu

2017-07-01

Virtual fluorescence emission difference microscopy (vFED) has been proposed recently to enhance the lateral resolution of confocal microscopy with a detector array, implemented by scanning a doughnut-shaped pattern. Theoretically, the resolution can be enhanced by around 1.3-fold compared with that in confocal microscopy. For further improvement of the resolving ability of vFED, a novel method is presented utilizing fluorescence saturation for super-resolution imaging, which we called saturated virtual fluorescence emission difference microscopy (svFED). With a point detector array, matched solid and hollow point spread functions (PSF) can be obtained by photon reassignment, and the difference results between them can be used to boost the transverse resolution. Results show that the diffraction barrier can be surpassed by at least 34% compared with that in vFED and the resolution is around 2-fold higher than that in confocal microscopy.

A practical approach to the diagnosis of systemic amyloidoses.

Science.gov (United States)

Fernández de Larrea, Carlos; Verga, Laura; Morbini, Patrizia; Klersy, Catherine; Lavatelli, Francesca; Foli, Andrea; Obici, Laura; Milani, Paolo; Capello, Gian Luca; Paulli, Marco; Palladini, Giovanni; Merlini, Giampaolo

2015-04-02

Accurate diagnosis of systemic amyloidosis is necessary both for assessing the prognosis and for delineating the appropriate treatment. It is based on histologic evidence of amyloid deposits and characterization of the amyloidogenic protein. We prospectively evaluated the diagnostic performance of immunoelectron microscopy (IEM) of abdominal fat aspirates from 745 consecutive patients with suspected systemic amyloidoses. All cases were extensively investigated with clinical and laboratory data, with a follow-up of at least 18 months. The 423 (56.8%) cases with confirmed systemic forms were used to estimate the diagnostic performance of IEM. Compared with Congo-red-based light microscopy, IEM was equally sensitive (75% to 80%) but significantly more specific (100% vs 80%; P 99% of the cases. IEM of abdominal fat aspirates is an effective tool in the routine diagnosis of systemic amyloidoses. © 2015 by The American Society of Hematology.

Simultaneous differential spinning disk fluorescence optical sectioning microscopy and nanomechanical mapping atomic force microscopy

International Nuclear Information System (INIS)

Miranda, Adelaide; De Beule, Pieter A. A.; Martins, Marco

2015-01-01

Combined microscopy techniques offer the life science research community a powerful tool to investigate complex biological systems and their interactions. Here, we present a new combined microscopy platform based on fluorescence optical sectioning microscopy through aperture correlation microscopy with a Differential Spinning Disk (DSD) and nanomechanical mapping with an Atomic Force Microscope (AFM). The illumination scheme of the DSD microscope unit, contrary to standard single or multi-point confocal microscopes, provides a time-independent illumination of the AFM cantilever. This enables a distortion-free simultaneous operation of fluorescence optical sectioning microscopy and atomic force microscopy with standard probes. In this context, we discuss sample heating due to AFM cantilever illumination with fluorescence excitation light. Integration of a DSD fluorescence optical sectioning unit with an AFM platform requires mitigation of mechanical noise transfer of the spinning disk. We identify and present two solutions to almost annul this noise in the AFM measurement process. The new combined microscopy platform is applied to the characterization of a DOPC/DOPS (4:1) lipid structures labelled with a lipophilic cationic indocarbocyanine dye deposited on a mica substrate

Simultaneous differential spinning disk fluorescence optical sectioning microscopy and nanomechanical mapping atomic force microscopy

Energy Technology Data Exchange (ETDEWEB)

Miranda, Adelaide; De Beule, Pieter A. A., E-mail: pieter.de-beule@inl.int [Applied Nano-Optics Laboratory, International Iberian Nanotechnology Laboratory, Avenida Mestre José Veiga, s/n, 4715-330 Braga (Portugal); Martins, Marco [Nano-ICs Group, International Iberian Nanotechnology Laboratory, Avenida Mestre José Veiga, s/n, 4715-330 Braga (Portugal)

2015-09-15

Combined microscopy techniques offer the life science research community a powerful tool to investigate complex biological systems and their interactions. Here, we present a new combined microscopy platform based on fluorescence optical sectioning microscopy through aperture correlation microscopy with a Differential Spinning Disk (DSD) and nanomechanical mapping with an Atomic Force Microscope (AFM). The illumination scheme of the DSD microscope unit, contrary to standard single or multi-point confocal microscopes, provides a time-independent illumination of the AFM cantilever. This enables a distortion-free simultaneous operation of fluorescence optical sectioning microscopy and atomic force microscopy with standard probes. In this context, we discuss sample heating due to AFM cantilever illumination with fluorescence excitation light. Integration of a DSD fluorescence optical sectioning unit with an AFM platform requires mitigation of mechanical noise transfer of the spinning disk. We identify and present two solutions to almost annul this noise in the AFM measurement process. The new combined microscopy platform is applied to the characterization of a DOPC/DOPS (4:1) lipid structures labelled with a lipophilic cationic indocarbocyanine dye deposited on a mica substrate.

Comparison between optical techniques and confocal microscopy for defect detection on thin wires

International Nuclear Information System (INIS)

Siegmann, Philip; Sanchez-Brea, Luis Miguel; Martinez-Anton, Juan Carlos; Bernabeu, Eusebio

2004-01-01

Conventional microscopy techniques, such as atomic force microscopy (AFM), scanning electron microscopy (SEM), and confocal microscopy (CM) are not suitable for on-line surface inspection of fine metallic wires. In the recent years, some optical techniques have been developed to be used for those tasks. However, they need a rigorous validation. In this work, we have used confocal microscopy to obtain the topography z(x,y) of wires with longitudinal defects, such as dielines. The topography has been used to predict the light scattered by the wire. These simulations have been compared with experimental results, showing a good agreement

Fluorescence (Multiwave) Confocal Microscopy.

Science.gov (United States)

Welzel, J; Kästle, Raphaela; Sattler, Elke C

2016-10-01

In addition to reflectance confocal microscopy, multiwave confocal microscopes with different laser wavelengths in combination with exogenous fluorophores allow fluorescence mode confocal microscopy in vivo and ex vivo. Fluorescence mode confocal microscopy improves the contrast between the epithelium and the surrounding soft tissue and allows the depiction of certain structures, like epithelial tumors, nerves, and glands. Copyright © 2016 Elsevier Inc. All rights reserved.

Overall major histocompatibility complex class I expression is not downregulated in cervix cancer, as detected by immunoelectron microscopy

NARCIS (Netherlands)

van Eijkeren, MA; Roovers, JP; Oorschot, [No Value; Geuze, HJ

2004-01-01

Downregulation of major histocompatibility complex (MHC) class I molecules in cervix cancer has been proposed as a mechanism for cancer cells to escape immunodetection. By means of light microscopic immunohistochemistry, it has been shown that in 20-70% of cervix cancers MHC class I is

Microscopy and Image Analysis.

Science.gov (United States)

McNamara, George; Difilippantonio, Michael; Ried, Thomas; Bieber, Frederick R

2017-07-11

This unit provides an overview of light microscopy, including objectives, light sources, filters, film, and color photography for fluorescence microscopy and fluorescence in situ hybridization (FISH). We believe there are excellent opportunities for cytogeneticists, pathologists, and other biomedical readers, to take advantage of specimen optical clearing techniques and expansion microscopy-we briefly point to these new opportunities. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Fusion of lens-free microscopy and mobile-phone microscopy images for high-color-accuracy and high-resolution pathology imaging

Science.gov (United States)

Zhang, Yibo; Wu, Yichen; Zhang, Yun; Ozcan, Aydogan

2017-03-01

Digital pathology and telepathology require imaging tools with high-throughput, high-resolution and accurate color reproduction. Lens-free on-chip microscopy based on digital in-line holography is a promising technique towards these needs, as it offers a wide field of view (FOV >20 mm2) and high resolution with a compact, low-cost and portable setup. Color imaging has been previously demonstrated by combining reconstructed images at three discrete wavelengths in the red, green and blue parts of the visible spectrum, i.e., the RGB combination method. However, this RGB combination method is subject to color distortions. To improve the color performance of lens-free microscopy for pathology imaging, here we present a wavelet-based color fusion imaging framework, termed "digital color fusion microscopy" (DCFM), which digitally fuses together a grayscale lens-free microscope image taken at a single wavelength and a low-resolution and low-magnification color-calibrated image taken by a lens-based microscope, which can simply be a mobile phone based cost-effective microscope. We show that the imaging results of an H&E stained breast cancer tissue slide with the DCFM technique come very close to a color-calibrated microscope using a 40x objective lens with 0.75 NA. Quantitative comparison showed 2-fold reduction in the mean color distance using the DCFM method compared to the RGB combination method, while also preserving the high-resolution features of the lens-free microscope. Due to the cost-effective and field-portable nature of both lens-free and mobile-phone microscopy techniques, their combination through the DCFM framework could be useful for digital pathology and telepathology applications, in low-resource and point-of-care settings.

International Multidisciplinary Microscopy Congress

CERN Document Server

Oral, Ahmet; Ozer, Mehmet; InterM; INTERM2013

2014-01-01

The International Multidisciplinary Microscopy Congress (INTERM2013) was organized on October 10-13, 2013. The aim of the congress was to bring together scientists from various branches to discuss the latest advances in the field of microscopy. The contents of the congress have been broadened to a more "interdisciplinary" scope, so as to allow all scientists working on related subjects to participate and present their work. These proceedings include 39 peer-reviewed technical papers, submitted by leading academic and research institutions from over 12 countries and representing some of the most cutting-edge research available. The 39 papers are grouped into the following sections: - Applications of Microscopy in the Physical Sciences - Applications of Microscopy in the Biological Sciences

Correlative microscopy of a carbide-free bainitic steel.

Science.gov (United States)

Hofer, Christina; Bliznuk, Vitaliy; Verdiere, An; Petrov, Roumen; Winkelhofer, Florian; Clemens, Helmut; Primig, Sophie

2016-02-01

In this work a carbide-free bainitic steel was examined by a novel correlative microscopy approach using transmission Kikuchi diffraction (TKD) and transmission electron microscopy (TEM). The individual microstructural constituents could be identified by TKD based on their different crystal structure for bainitic ferrite and retained austenite and by image quality for the martensite-austenite (M-A) constituent. Subsequently, the same area was investigated in the TEM and a good match of these two techniques regarding the identification of the area position and crystal orientation could be proven. Additionally, the M-A constituent was examined in the TEM for the first time after preceded unambiguous identification using a correlative microscopy approach. The selected area diffraction pattern showed satellites around the main reflexes which might indicate a structural modulation. Copyright © 2015 Elsevier Ltd. All rights reserved.

Laminin in the anterior pituitary gland of the rat. Laminin in the gonadotrophic cells correlates with their functional state

DEFF Research Database (Denmark)

Holck, S; Albrechtsen, R; Wewer, U M

1987-01-01

The distribution pattern of laminin in the rat anterior pituitary gland under physiological and hormonally altered conditions was studied immunohistochemically. Intense immunoreactivity of the capillaries and of the basement membranes surrounding parenchymal cells was found. Five to 10......% of the parenchymal cells of normal adult rat pituitary gland exhibited also intense positive cytoplasmic staining. These were identified as gonadotrophic cells on the basis of their topographic distribution and typical 700-nm light bodies. By immunoelectron microscopy it was shown that the light bodies contain...... laminin and the number of light bodies reflects the hormonal activity of the gonadotrophic cells of the rat pituitary gland....

The transferrin receptor of Actinobacillus pleuropneumoniae: Quantitation of expression and structural characterization using a peptide-specific monoclonal antibody

DEFF Research Database (Denmark)

Bøg, Yang S.; Andresen, Lars Ole; Bastholm, L.

2001-01-01

transferrin. This complex was studied using a monoclonal antibody (Mab 1.48) raised against a synthetic peptide corresponding to a hydrophilic domain of Tbp2 common to several A. pp serotypes. The antibody reacted specifically with a 60-70 kDa Tbp2-antigen found in all serotypes of A. pp obtained from iron...... expressing Tbp2 and in wild type A. pp grown under iron restricted conditions. The subcellular location of Tbp2 in A. pp was studied by immunoelectron microscopy using the Mab 1.48. Interestingly, all antibody binding was found inside the A. pp cells, while Tbp2 expressed in recombinant E. coli was found...

Resolving amorphous solid-liquid interfaces by atomic force microscopy

International Nuclear Information System (INIS)

Burson, Kristen M.; Gura, Leonard; Kell, Burkhard; Büchner, Christin; Lewandowski, Adrian L.; Heyde, Markus; Freund, Hans-Joachim

2016-01-01

Recent advancements in liquid atomic force microscopy make it an ideal technique for probing the structure of solid-liquid interfaces. Here, we present a structural study of a two-dimensional amorphous silica bilayer immersed in an aqueous solution utilizing liquid atomic force microscopy with sub-nanometer resolution. Structures show good agreement with atomically resolved ultra-high vacuum scanning tunneling microscopy images obtained on the same sample system, owing to the structural stability of the silica bilayer and the imaging clarity from the two-dimensional sample system. Pair distance histograms of ring center positions are utilized to develop quantitative metrics for structural comparison, and the physical origin of pair distance histogram peaks is addressed by direct assessment of real space structures.

Polarization contrast in photon scanning tunnelling microscopy combined with atomic force microscopy

NARCIS (Netherlands)

Propstra, K.; Propstra, K.; van Hulst, N.F.

1995-01-01

Photon scanning tunnelling microscopy combined with atomic force microscopy allows simultaneous acquisition and direct comparison of optical and topographical images, both with a lateral resolution of about 30 nm, far beyond the optical diffraction limit. The probe consists of a modified

CARS microscopy for imaging

International Nuclear Information System (INIS)

Arzumanyan Grigory; Voskanyan Karine

2013-01-01

Optical microscopy grows in its importance with the development of modern nanotechnology, biotechnology, methods of diagnostics and treatment of most dangerous diseases for mankind. There are several important goals of optical microscopy for biomedical studies among which the next three may be distinguished: fast imaging with high lateral spatial resolution, 3-D sectioning capability and high contrast for chemical selectivity. To meet these specific requirements, various types of both linear and nonlinear optical microscopy were elaborated. (authors)

Tapping mode atomic force microscopy in liquid

NARCIS (Netherlands)

Putman, Constant A.J.; Putman, C.A.J.; van der Werf, Kees; de Grooth, B.G.; van Hulst, N.F.; Greve, Jan

1994-01-01

We show that standard silicon nitride cantilevers can be used for tapping mode atomic force microscopy (AFM) in air, provided that the energy of the oscillating cantilever is sufficiently high to overcome the adhesion of the water layer. The same cantilevers are successfully used for tapping mode

Concepts in Light Microscopy of Viruses

Science.gov (United States)

Witte, Robert; Georgi, Fanny

2018-01-01

Viruses threaten humans, livestock, and plants, and are difficult to combat. Imaging of viruses by light microscopy is key to uncover the nature of known and emerging viruses in the quest for finding new ways to treat viral disease and deepening the understanding of virus–host interactions. Here, we provide an overview of recent technology for imaging cells and viruses by light microscopy, in particular fluorescence microscopy in static and live-cell modes. The review lays out guidelines for how novel fluorescent chemical probes and proteins can be used in light microscopy to illuminate cells, and how they can be used to study virus infections. We discuss advantages and opportunities of confocal and multi-photon microscopy, selective plane illumination microscopy, and super-resolution microscopy. We emphasize the prevalent concepts in image processing and data analyses, and provide an outlook into label-free digital holographic microscopy for virus research. PMID:29670029

Orexin neurons receive glycinergic innervations.

Directory of Open Access Journals (Sweden)

Mari Hondo

Full Text Available Glycine, a nonessential amino-acid that acts as an inhibitory neurotransmitter in the central nervous system, is currently used as a dietary supplement to improve the quality of sleep, but its mechanism of action is poorly understood. We confirmed the effects of glycine on sleep/wakefulness behavior in mice when administered peripherally. Glycine administration increased non-rapid eye movement (NREM sleep time and decreased the amount and mean episode duration of wakefulness when administered in the dark period. Since peripheral administration of glycine induced fragmentation of sleep/wakefulness states, which is a characteristic of orexin deficiency, we examined the effects of glycine on orexin neurons. The number of Fos-positive orexin neurons markedly decreased after intraperitoneal administration of glycine to mice. To examine whether glycine acts directly on orexin neurons, we examined the effects of glycine on orexin neurons by patch-clamp electrophysiology. Glycine directly induced hyperpolarization and cessation of firing of orexin neurons. These responses were inhibited by a specific glycine receptor antagonist, strychnine. Triple-labeling immunofluorescent analysis showed close apposition of glycine transporter 2 (GlyT2-immunoreactive glycinergic fibers onto orexin-immunoreactive neurons. Immunoelectron microscopic analysis revealed that GlyT2-immunoreactive terminals made symmetrical synaptic contacts with somata and dendrites of orexin neurons. Double-labeling immunoelectron microscopy demonstrated that glycine receptor alpha subunits were localized in the postsynaptic membrane of symmetrical inhibitory synapses on orexin neurons. Considering the importance of glycinergic regulation during REM sleep, our observations suggest that glycine injection might affect the activity of orexin neurons, and that glycinergic inhibition of orexin neurons might play a role in physiological sleep regulation.

Bridging fluorescence microscopy and electron microscopy

NARCIS (Netherlands)

Giepmans, Ben N. G.

Development of new fluorescent probes and fluorescence microscopes has led to new ways to study cell biology. With the emergence of specialized microscopy units at most universities and research centers, the use of these techniques is well within reach for a broad research community. A major

Validation of Digital Microscopy Compared With Light Microscopy for the Diagnosis of Canine Cutaneous Tumors.

Science.gov (United States)

Bertram, Christof A; Gurtner, Corinne; Dettwiler, Martina; Kershaw, Olivia; Dietert, Kristina; Pieper, Laura; Pischon, Hannah; Gruber, Achim D; Klopfleisch, Robert

2018-07-01

Integration of new technologies, such as digital microscopy, into a highly standardized laboratory routine requires the validation of its performance in terms of reliability, specificity, and sensitivity. However, a validation study of digital microscopy is currently lacking in veterinary pathology. The aim of the current study was to validate the usability of digital microscopy in terms of diagnostic accuracy, speed, and confidence for diagnosing and differentiating common canine cutaneous tumor types and to compare it to classical light microscopy. Therefore, 80 histologic sections including 17 different skin tumor types were examined twice as glass slides and twice as digital whole-slide images by 6 pathologists with different levels of experience at 4 time points. Comparison of both methods found digital microscopy to be noninferior for differentiating individual tumor types within the category epithelial and mesenchymal tumors, but diagnostic concordance was slightly lower for differentiating individual round cell tumor types by digital microscopy. In addition, digital microscopy was associated with significantly shorter diagnostic time, but diagnostic confidence was lower and technical quality was considered inferior for whole-slide images compared with glass slides. Of note, diagnostic performance for whole-slide images scanned at 200× magnification was noninferior in diagnostic performance for slides scanned at 400×. In conclusion, digital microscopy differs only minimally from light microscopy in few aspects of diagnostic performance and overall appears adequate for the diagnosis of individual canine cutaneous tumors with minor limitations for differentiating individual round cell tumor types and grading of mast cell tumors.

Exploring neural cell dynamics with digital holographic microscopy

KAUST Repository

Marquet, Pierre; Depeursinge, Christian D.; Magistretti, Pierre J.

2013-01-01

In this review, we summarize how the new concept of digital optics applied to the field of holographic microscopy has allowed the development of a reliable and flexible digital holographic quantitative phase microscopy (DH-QPM) technique at the nanoscale particularly suitable for cell imaging. Particular emphasis is placed on the original biological ormation provided by the quantitative phase signal. We present the most relevant DH-QPM applications in the field of cell biology, including automated cell counts, recognition, classification, three-dimensional tracking, discrimination between physiological and pathophysiological states, and the study of cell membrane fluctuations at the nanoscale. In the last part, original results show how DH-QPM can address two important issues in the field of neurobiology, namely, multiple-site optical recording of neuronal activity and noninvasive visualization of dendritic spine dynamics resulting from a full digital holographic microscopy tomographic approach. Copyright © 2013 by Annual Reviews.

Exploring neural cell dynamics with digital holographic microscopy

KAUST Repository

Marquet, Pierre

2013-07-11

In this review, we summarize how the new concept of digital optics applied to the field of holographic microscopy has allowed the development of a reliable and flexible digital holographic quantitative phase microscopy (DH-QPM) technique at the nanoscale particularly suitable for cell imaging. Particular emphasis is placed on the original biological ormation provided by the quantitative phase signal. We present the most relevant DH-QPM applications in the field of cell biology, including automated cell counts, recognition, classification, three-dimensional tracking, discrimination between physiological and pathophysiological states, and the study of cell membrane fluctuations at the nanoscale. In the last part, original results show how DH-QPM can address two important issues in the field of neurobiology, namely, multiple-site optical recording of neuronal activity and noninvasive visualization of dendritic spine dynamics resulting from a full digital holographic microscopy tomographic approach. Copyright © 2013 by Annual Reviews.

Fast Calcium Imaging with Optical Sectioning via HiLo Microscopy.

Science.gov (United States)

Lauterbach, Marcel A; Ronzitti, Emiliano; Sternberg, Jenna R; Wyart, Claire; Emiliani, Valentina

2015-01-01

Imaging intracellular calcium concentration via reporters that change their fluorescence properties upon binding of calcium, referred to as calcium imaging, has revolutionized our way to probe neuronal activity non-invasively. To reach neurons densely located deep in the tissue, optical sectioning at high rate of acquisition is necessary but difficult to achieve in a cost effective manner. Here we implement an accessible solution relying on HiLo microscopy to provide robust optical sectioning with a high frame rate in vivo. We show that large calcium signals can be recorded from dense neuronal populations at high acquisition rates. We quantify the optical sectioning capabilities and demonstrate the benefits of HiLo microscopy compared to wide-field microscopy for calcium imaging and 3D reconstruction. We apply HiLo microscopy to functional calcium imaging at 100 frames per second deep in biological tissues. This approach enables us to discriminate neuronal activity of motor neurons from different depths in the spinal cord of zebrafish embryos. We observe distinct time courses of calcium signals in somata and axons. We show that our method enables to remove large fluctuations of the background fluorescence. All together our setup can be implemented to provide efficient optical sectioning in vivo at low cost on a wide range of existing microscopes.

French Society of Microscopy, 10. conference

International Nuclear Information System (INIS)

Thibault-Penisson, J.; Cremer, Ch.; Susini, J.; Kirklanda, A.I.; Rigneault, H.; Renault, O.; Bailly, A.; Zagonel, L.F.; Barrett, N.; Bogner, A.; Gauthier, C.; Jouneau, P.H.; Thollet, G.; Fuchs, G.; Basset, D.; Deconihout, B.; Vurpillot, F.; Vella, A.; Matthieu, G.; Cadel, E.; Bostel, A.; Blavette, D.; Baumeister, W.; Usson, Y.; Zaefferer, St.; Laffont, L.; Weyland, M.; Thomas, J.M.; Midgley, P.; Benlekbir, S.; Epicier, Th.; Diop, B.N.; Roux, St.; Ou, M.; Perriat, P.; Bausach, M.; Aouine, M.; Berhault, G.; Idrissi, H.; Cottevieille, M.; Jonic, S.; Larquet, E.; Svergun, D.; Vannoni, M.A.; Boisset, N.; Ersena, O.; Werckmann, J.; Ulhaq, C.; Hirlimann, Ch.; Tihay, F.; Cuong, Pham-Huu; Crucifix, C.; Schultz, P.; Jornsanoha, P.; Thollet, G.; Masenelli-Varlot, K.; Gauthier, C.; Ludwig, W.; King, A.; Johnson, G.; Gonzalves-Hoennicke, M.; Reischig, P.; Messaoudi, C.; Ibrahim, R.; Marco, S.; Klie, R.F.; Zhao, Y.; Yang, G.; Zhu, Y.; Hue, F.; Hytch, M.; Hartmann, J.M.; Bogumilowicz, Y.; Claverie, A.; Klein, H.; Alloyeau, D.; Ricolleau, C.; Langlois, C.; Le Bouar, Y.; Loiseau, A.; Colliex, C.; Stephan, O.; Kociak, M.; Tence, M.; Gloter, A.; Imhoff, D.; Walls, M.; Nelayah, J.; March, K.; Couillard, M.; Ailliot, C.; Bertin, F.; Cooper, D.; Rivallin, P.; Dumelie, N.; Benhayoune, H.; Balossier, G.; Cheynet, M.; Pokrant, S.; Tichelaar, F.; Rouviere, J.L.; Cooper, D.; Truche, R.; Chabli, A.; Debili, M.Y.; Houdellier, F.; Warot-Fonrose, B.; Hytch, M.J.; Snoeck, E.; Calmels, L.; Serin, V.; Schattschneider, P.; Jacob, D.; Cordier, P.

2007-01-01

This document gathers the resumes of some of the presentations made at this conference whose aim was to present the last developments and achievements of the 3 complementary microscopies: optical microscopy, electron microscopy and X-ray microscopy. The contributions have been organized around the following 12 topics: 1) new technical developments, 2) 3-dimensional imaging, 3) quantitative microscopy, 4) technical progress in photon microscopy, 5) synchrotron radiation, 6) measurements of patterns, deformations and strains, 7) materials for energy and transports, 8) nano-structures, 9) virus: structure and infection mechanisms, 10) 3-dimensional imaging for molecules, cells and cellular tissues, 11) nano-particles and colloids, and 12) liquid crystals

Fabrication and atomic force microscopy/friction force microscopy (AFM/FFM) studies of polyacrylamide-carbon nanotubes (PAM-CNTs) copolymer thin films

International Nuclear Information System (INIS)

Li Xuefeng; Guan Wenchao; Yan Haibiao; Huang Lan

2004-01-01

A novel polyacrylamide-carbon nanotubes (PAM-CNTs) copolymer has been prepared by ultraviolet radiation initiated polymerization. The PAM-CNTs copolymer was characterized by the instruments of Fourier transform infrared spectroscopy, UV-vis absorbance spectra, fluorescence spectra and transmission electron microscope. The morphology and microtribological properties of PAM-CNTs thin films on mica were investigated by atomic force microscopy/friction force microscopy (AFM/FFM). The friction of the films was stable with the change of applied load and the friction coefficient decreased significantly as the CNTs addition. The results show that the rigid rod-like CNTs in polymer would enhance load-bearing and anti-wear properties of the thin films

Microscopy techniques in flavivirus research.

Science.gov (United States)

Chong, Mun Keat; Chua, Anthony Jin Shun; Tan, Terence Tze Tong; Tan, Suat Hoon; Ng, Mah Lee

2014-04-01

The Flavivirus genus is composed of many medically important viruses that cause high morbidity and mortality, which include Dengue and West Nile viruses. Various molecular and biochemical techniques have been developed in the endeavour to study flaviviruses. However, microscopy techniques still have irreplaceable roles in the identification of novel virus pathogens and characterization of morphological changes in virus-infected cells. Fluorescence microscopy contributes greatly in understanding the fundamental viral protein localizations and virus-host protein interactions during infection. Electron microscopy remains the gold standard for visualizing ultra-structural features of virus particles and infected cells. New imaging techniques and combinatory applications are continuously being developed to push the limit of resolution and extract more quantitative data. Currently, correlative live cell imaging and high resolution three-dimensional imaging have already been achieved through the tandem use of optical and electron microscopy in analyzing biological specimens. Microscopy techniques are also used to measure protein binding affinities and determine the mobility pattern of proteins in cells. This chapter will consolidate on the applications of various well-established microscopy techniques in flavivirus research, and discuss how recently developed microscopy techniques can potentially help advance our understanding in these membrane viruses. Copyright © 2013 Elsevier Ltd. All rights reserved.

Localization of adenovirus morphogenesis players, together with visualization of assembly intermediates and failed products, favor a model where assembly and packaging occur concurrently at the periphery of the replication center.

Directory of Open Access Journals (Sweden)

Gabriela N Condezo

2017-04-01

Full Text Available Adenovirus (AdV morphogenesis is a complex process, many aspects of which remain unclear. In particular, it is not settled where in the nucleus assembly and packaging occur, and whether these processes occur in a sequential or a concerted manner. Here we use immunofluorescence and immunoelectron microscopy (immunoEM to trace packaging factors and structural proteins at late times post infection by either wildtype virus or a delayed packaging mutant. We show that representatives of all assembly factors are present in the previously recognized peripheral replicative zone, which therefore is the AdV assembly factory. Assembly intermediates and abortive products observed in this region favor a concurrent assembly and packaging model comprising two pathways, one for capsid proteins and another one for core components. Only when both pathways are coupled by correct interaction between packaging proteins and the genome is the viral particle produced. Decoupling generates accumulation of empty capsids and unpackaged cores.

Subcellular localization of hepatitis E virus (HEV) replicase

International Nuclear Information System (INIS)

Rehman, Shagufta; Kapur, Neeraj; Durgapal, Hemlata; Panda, Subrat Kumar

2008-01-01

Hepatitis E virus (HEV) is a hepatotropic virus with a single sense-strand RNA genome of ∼ 7.2 kb in length. Details of the intracellular site of HEV replication can pave further understanding of HEV biology. In-frame fusion construct of functionally active replicase-enhanced green fluorescent protein (EGFP) gene was made in eukaryotic expression vector. The functionality of replicase-EGFP fusion protein was established by its ability to synthesize negative-strand viral RNA in vivo, by strand-specific anchored RT-PCR and molecular beacon binding. Subcellular co-localization was carried out using organelle specific fluorophores and by immuno-electron microscopy. Fluorescence Resonance Energy Transfer (FRET) demonstrated the interaction of this protein with the 3' end of HEV genome. The results show localization of replicase on the endoplasmic reticulum membranes. The protein regions responsible for membrane localization was predicted and identified by use of deletion mutants. Endoplasmic reticulum was identified as the site of replicase localization and possible site of replication

Localization of three human polypeptide GalNAc-transferases in HeLa cells suggests initiation of O-linked glycosylation throughout the Golgi apparatus

DEFF Research Database (Denmark)

Röttger, S; White, J; Wandall, H H

1998-01-01

O-glycosylation of proteins is initiated by a family of UDP-N-acetylgalactosamine:polypeptide N-acetylgalactos-aminyltransferases (GalNAc-T). In this study, we have localized endogenous and epitope-tagged human GalNAc-T1, -T2 and -T3 to the Golgi apparatus in HeLa cells by subcellular fractionation......, immunofluorescence and immunoelectron microscopy. We show that all three GalNAc-transferases are concentrated about tenfold in Golgi stacks over Golgi associated tubular-vesicular membrane structures. Surprisingly, we find that GalNAc-T1, -T2 and -T3 are present throughout the Golgi stack suggesting that initiation...... of O-glycosylation may not be restricted to the cis Golgi, but occur at multiple sites within the Golgi apparatus. GalNAc-T1 distributes evenly across the Golgi stack whereas GalNAc-T2 and -T3 reside preferentially on the trans side and in the medial part of the Golgi stack, respectively. Moreover, we...

Towards the use of protein A-tagged gold nanoparticles for signal amplification of electrochemical immunosensors in virus detection

International Nuclear Information System (INIS)

Huy Tran, Quang; Thuy Nguyen, Thanh; Chung Pham, Van; Hong Hanh Nguyen, Thi; Tuan Mai, Anh

2012-01-01

In this paper we represent a study on the potential use of protein A-tagged gold nanoparticles applied for signal amplification of electrochemical immunosensors. Gold nanoparticles (GNPs) were synthesized by the chemical reduction of tetrachloroauric (III) acid trihydrate using sodium ascorbate, and then tagged with protein A (PrA) via ultracentrifugation. UV-Vis spectroscopy and transmission electron microscopy were used to verify the characteristics of formed GNPs/PrA complex. The analyzed results indicate that GNPs were found spherically, homogeneously, and with an average diameter of about 10 nm. Immunoelectron microscopy was then used to investigate the bioactivity of the GNPs/PrA complex in solution by the effective binding of GNPs to viral particles. Scanning electron and fluorescence microscopies were also used to investigate the distribution and the bioactivity of the GNPs/PrA complex on the surface of the interdigitated sensor. Consequently, this study provided some assumptions of the potential application of protein A-tagged gold nanoparticles for signal amplification of electrochemical immunosensors in virus detection from clinical samples

Confocal Raman Microscopy

CERN Document Server

Dieing, Thomas; Toporski, Jan

2011-01-01

Confocal Raman Microscopy is a relatively new technique that allows chemical imaging without specific sample preparation. By integrating a sensitive Raman spectrometer within a state-of-the-art microscope, Raman microscopy with a spatial resolution down to 200nm laterally and 500nm vertically can be achieved using visible light excitation. Recent developments in detector and computer technology as well as optimized instrument design have reduced integration times of Raman spectra by orders of magnitude, so that complete images consisting of tens of thousands of Raman spectra can be acquired in seconds or minutes rather than hours, which used to be standard just one decade ago. The purpose of this book is to provide the reader a comprehensive overview of the rapidly developing field of Confocal Raman Microscopy and its applications.

Fluorescence confocal microscopy for pathologists.

Science.gov (United States)

Ragazzi, Moira; Piana, Simonetta; Longo, Caterina; Castagnetti, Fabio; Foroni, Monica; Ferrari, Guglielmo; Gardini, Giorgio; Pellacani, Giovanni

2014-03-01

Confocal microscopy is a non-invasive method of optical imaging that may provide microscopic images of untreated tissue that correspond almost perfectly to hematoxylin- and eosin-stained slides. Nowadays, following two confocal imaging systems are available: (1) reflectance confocal microscopy, based on the natural differences in refractive indices of subcellular structures within the tissues; (2) fluorescence confocal microscopy, based on the use of fluorochromes, such as acridine orange, to increase the contrast epithelium-stroma. In clinical practice to date, confocal microscopy has been used with the goal of obviating the need for excision biopsies, thereby reducing the need for pathological examination. The aim of our study was to test fluorescence confocal microscopy on different types of surgical specimens, specifically breast, lymph node, thyroid, and colon. The confocal images were correlated to the corresponding histological sections in order to provide a morphologic parallel and to highlight current limitations and possible applications of this technology for surgical pathology practice. As a result, neoplastic tissues were easily distinguishable from normal structures and reactive processes such as fibrosis; the use of fluorescence enhanced contrast and image quality in confocal microscopy without compromising final histologic evaluation. Finally, the fluorescence confocal microscopy images of the adipose tissue were as accurate as those of conventional histology and were devoid of the frozen-section-related artefacts that can compromise intraoperative evaluation. Despite some limitations mainly related to black/white images, which require training in imaging interpretation, this study confirms that fluorescence confocal microscopy may represent an alternative to frozen sections in the assessment of margin status in selected settings or when the conservation of the specimen is crucial. This is the first study to employ fluorescent confocal microscopy on

Electron microscopy for Engineers

International Nuclear Information System (INIS)

Jones, I P

2009-01-01

This paper reviews the application of (mainly) Transmission Electron Microscopy (TEM) in an engineering context. The first two sections are TEM and chemical in nature; the final three sections are more general and include aspects of Scanning Electron Microscopy (SEM).

Qualitative doping area characterization of SONOS transistor utilizing scanning capacitance microscopy (SCM) and scanning spread resistance microscopy (SSRM)

International Nuclear Information System (INIS)

Heo, Jinhee; Kim, Deoksu; Kim, Chung woo; Chung, Ilsub

2005-01-01

Continuous shrinkage in the memory devices demands further understanding about the doping concentration variations at shallow junction and channel region. Scanning capacitance microscopy (SCM) and scanning spread resistance microscopy (SSRM) can provide reliable information about the electrical and physical junction structure simultaneously. In this work, we attempt to visualize the doping concentration variations of split-gate structure silicon-oxide-nitride-oxide-silicon (SONOS) transistor with thin oxide-nitride-oxide (ONO; 4/7/11 nm). From SCM image, we could identify the source and drain region, which have different doping concentrations from that at channel region. In addition, a gate oxide layer and a depletion region were also identified. Similar results were obtained using SSRM. However, SSRM shows a better resolution, in particular, for highly doped region. For this experiment, the cross-sectional sample has been prepared using focused ion beam (FIB) and hand-polishing method. The results show that SCM and SSRM are very useful methods to analyze the doping profile near the junction as well as the channel

CNNs for electron microscopy segmentation

OpenAIRE

García-Amorena García, Pablo

2013-01-01

In the framework of Biomedicine, mitochondria are known to play an important role in neural function. Recent studies show mitochondrial morphology to be crucial to cellular physiology and synaptic function, and a link between mitochondrial defects and neuro-degenerative diseases is strongly suspected. Electron microscopy (EM), with its very high resolution in all three directions, is one of the key tools to look more closely into these tissues, but the huge amounts of data it produces m...

Superresolution size determination in fluorescence microscopy: A comparison between spatially modulated illumination and confocal laser scanning microscopy

International Nuclear Information System (INIS)

Spoeri, Udo; Failla, Antonio Virgilio; Cremer, Christoph

2004-01-01

Recently developed far field light optical methods are a powerful tool to analyze biological nanostructures and their dynamics, in particular including the interior of three-dimensionally conserved cells. In this article, the recently described method of spatially modulated illumination (SMI) microscopy has been further extended to the online determination of the extension of small, subwavelength sized, fluorescent objects (nanosizing). Using fluorescence excitation with 488 nm, the determination of fluorescent labeled object diameters down to 40 nm corresponding to about 1/12th of the wavelength used for one-photon excitation could be shown. The results of the SMI nanosizing procedure for a detailed, systematic variation of the object diameter are presented together with a fast algorithm for online size evaluation. In addition, we show a direct comparison of the diameter of 'colocalization volumes' between SMI nanosizing and conventional confocal laser scanning microscopy

Low Dimensional Representation of Fisher Vectors for Microscopy Image Classification.

Science.gov (United States)

Song, Yang; Li, Qing; Huang, Heng; Feng, Dagan; Chen, Mei; Cai, Weidong

2017-08-01

Microscopy image classification is important in various biomedical applications, such as cancer subtype identification, and protein localization for high content screening. To achieve automated and effective microscopy image classification, the representative and discriminative capability of image feature descriptors is essential. To this end, in this paper, we propose a new feature representation algorithm to facilitate automated microscopy image classification. In particular, we incorporate Fisher vector (FV) encoding with multiple types of local features that are handcrafted or learned, and we design a separation-guided dimension reduction method to reduce the descriptor dimension while increasing its discriminative capability. Our method is evaluated on four publicly available microscopy image data sets of different imaging types and applications, including the UCSB breast cancer data set, MICCAI 2015 CBTC challenge data set, and IICBU malignant lymphoma, and RNAi data sets. Our experimental results demonstrate the advantage of the proposed low-dimensional FV representation, showing consistent performance improvement over the existing state of the art and the commonly used dimension reduction techniques.

Induction of morphological changes in death-induced cancer cells monitored by holographic microscopy.

Science.gov (United States)

El-Schich, Zahra; Mölder, Anna; Tassidis, Helena; Härkönen, Pirkko; Falck Miniotis, Maria; Gjörloff Wingren, Anette

2015-03-01

We are using the label-free technique of holographic microscopy to analyze cellular parameters including cell number, confluence, cellular volume and area directly in the cell culture environment. We show that death-induced cells can be distinguished from untreated counterparts by the use of holographic microscopy, and we demonstrate its capability for cell death assessment. Morphological analysis of two representative cell lines (L929 and DU145) was performed in the culture flasks without any prior cell detachment. The two cell lines were treated with the anti-tumour agent etoposide for 1-3days. Measurements by holographic microscopy showed significant differences in average cell number, confluence, volume and area when comparing etoposide-treated with untreated cells. The cell volume of the treated cell lines was initially increased at early time-points. By time, cells decreased in volume, especially when treated with high doses of etoposide. In conclusion, we have shown that holographic microscopy allows label-free and completely non-invasive morphological measurements of cell growth, viability and death. Future applications could include real-time monitoring of these holographic microscopy parameters in cells in response to clinically relevant compounds. Copyright © 2015 Elsevier Inc. All rights reserved.

Fourier plane imaging microscopy

Energy Technology Data Exchange (ETDEWEB)

Dominguez, Daniel, E-mail: daniel.dominguez@ttu.edu; Peralta, Luis Grave de [Department of Physics, Texas Tech University, Lubbock, Texas 79409 (United States); Nano Tech Center, Texas Tech University, Lubbock, Texas 79409 (United States); Alharbi, Nouf; Alhusain, Mdhaoui [Department of Physics, Texas Tech University, Lubbock, Texas 79409 (United States); Bernussi, Ayrton A. [Nano Tech Center, Texas Tech University, Lubbock, Texas 79409 (United States); Department of Electrical and Computer Engineering, Texas Tech University, Lubbock, Texas 79409 (United States)

2014-09-14

We show how the image of an unresolved photonic crystal can be reconstructed using a single Fourier plane (FP) image obtained with a second camera that was added to a traditional compound microscope. We discuss how Fourier plane imaging microscopy is an application of a remarkable property of the obtained FP images: they contain more information about the photonic crystals than the images recorded by the camera commonly placed at the real plane of the microscope. We argue that the experimental results support the hypothesis that surface waves, contributing to enhanced resolution abilities, were optically excited in the studied photonic crystals.

Polarized Light Microscopy

Science.gov (United States)

Frandsen, Athela F.

2016-01-01

Polarized light microscopy (PLM) is a technique which employs the use of polarizing filters to obtain substantial optical property information about the material which is being observed. This information can be combined with other microscopy techniques to confirm or elucidate the identity of an unknown material, determine whether a particular contaminant is present (as with asbestos analysis), or to provide important information that can be used to refine a manufacturing or chemical process. PLM was the major microscopy technique in use for identification of materials for nearly a century since its introduction in 1834 by William Fox Talbot, as other techniques such as SEM (Scanning Electron Microscopy), FTIR (Fourier Transform Infrared spectroscopy), XPD (X-ray Powder Diffraction), and TEM (Transmission Electron Microscopy) had not yet been developed. Today, it is still the only technique approved by the Environmental Protection Agency (EPA) for asbestos analysis, and is often the technique first applied for identification of unknown materials. PLM uses different configurations in order to determine different material properties. With each configuration additional clues can be gathered, leading to a conclusion of material identity. With no polarizing filter, the microscope can be used just as a stereo optical microscope, and view qualities such as morphology, size, and number of phases. With a single polarizing filter (single polars), additional properties can be established, such as pleochroism, individual refractive indices, and dispersion staining. With two polarizing filters (crossed polars), even more can be deduced: isotropy vs. anisotropy, extinction angle, birefringence/degree of birefringence, sign of elongation, and anomalous polarization colors, among others. With the use of PLM many of these properties can be determined in a matter of seconds, even for those who are not highly trained. McCrone, a leader in the field of polarized light microscopy, often

Reflectance Confocal Microscopy in Lentigo Maligna.

Science.gov (United States)

Gamo, R; Pampín, A; Floristán, U

2016-12-01

Lentigo maligna is the most common type of facial melanoma. Diagnosis is complicated, however, as it shares clinical and dermoscopic characteristics with other cutaneous lesions of the face. Reflectance confocal microscopy is an imaging technique that permits the visualization of characteristic features of lentigo maligna. These include a disrupted honeycomb pattern and pagetoid cells with a tendency to show folliculotropism. These cells typically have a dendritic morphology, although they may also appear as round cells measuring over 20μm with atypical nuclei. Poorly defined dermal papillae and atypical cells may be seen at the dermal-epidermal junction and can form bridges resembling mitochondrial structures. Other characteristic findings include junctional swelling with atypical cells located around the follicles, resembling caput medusae. Reflectance confocal microscopy is a very useful tool for diagnosing lentigo maligna. Copyright © 2016 AEDV. Publicado por Elsevier España, S.L.U. All rights reserved.

Lipid droplets, perilipins and cytokeratins--unravelled liaisons in epithelium-derived cells.

Directory of Open Access Journals (Sweden)

Hans Heid

Full Text Available Lipid droplets (LDs are spherical accumulations of apolar lipids and other hydrophobic substances and are generally surrounded by a thin cortical layer of specific amphiphilic proteins (APs. These APs segregate the LDs from the mostly polar components of the cytoplasm. We have studied LDs in epithelium-derived cell cultures and in particular characterized proteins from the perilipin (PLIN gene family - in mammals consisting of the proteins Perilipin, Adipophilin, TIP47, S3-12 and MLDP/OXPAT (PLIN 1-5. Using a large number of newly generated and highly specific mono- and polyclonal antibodies specific for individual APs, and using improved LD isolation methods, we have enriched and characterized APs in greater detail and purity. The majority of lipid-AP complexes could be obtained in the top layer fractions of density gradient centrifugation separations of cultured cells, but APs could also be detected in other fractions within such separations. The differently sized LD complexes were analyzed using various biochemical methods and mass spectrometry as well as immunofluorescence and electron- in particular immunoelectron-microscopy. Moreover, by immunoprecipitation, protein-protein binding assays and by immunoelectron microscopy we identified a direct linkage between LD-binding proteins and the intermediate-sized filaments (IF cytokeratins 8 and 18 (also designated as keratins K8 and K18. Specifically, in gradient fractions of higher density supposedly containing small LDs, we received as co-precipitations cytidylyl-, palmitoyl- and cholesterol transferases and other specific enzymes involved in lipid metabolism. So far, common proteomic studies have used LDs from top layer fractions only and did not report on these transferases and other enzymes. In addition to findings of short alternating hydrophobic/hydrophilic segments within the PLIN protein family, we propose and discuss a model for the interaction of LD-coating APs with IF proteins.

Investigation of non-collinear spin states with scanning tunneling microscopy.

Science.gov (United States)

Wulfhekel, W; Gao, C L

2010-03-05

Most ferromagnetic and antiferromagnetic substances show a simple collinear arrangement of the local spins. Under certain circumstances, however, the spin configuration is non-collinear. Scanning tunneling microscopy with its potential atomic resolution is an ideal tool for investigating these complex spin structures. Non-collinearity can be due to topological frustration of the exchange interaction, due to relativistic spin-orbit coupling or can be found in excited states. Examples for all three cases are given, illustrating the capabilities of spin-polarized scanning tunneling microscopy.

Coherent Raman scattering microscopy for label-free imaging of live amphioxus

Science.gov (United States)

Yu, Zhilong; Chen, Tao; Zhang, Xiannian; Shen, Jie; Chen, Junyuan; Huang, Yanyi

2012-03-01

The existence of notochord distinguishes chordates from other phyla. Amphioxus is the only animal that keeps notochord during the whole life. Notochord is a unique organ for amphioxus, with its vertically arranged muscular notochordal plates, which is different from notochords in embryos of other chordates. We use stimulated Raman scattering (SRS) microscopy as a non-invasive technique to image the chemical components in amphioxus notochord. SRS provides chemical specificity as spontaneous Raman does and offers a higher sensitivity for fast acquisition. Unlike coherent anti- Stokes Raman scattering (CARS) microscopy, SRS microscopy doesn't have non-resonant background and can better differentiate different components in the specimen. We verify that the notochord is a protein-rich organ, which agrees well with the result of conventional staining methods. Detailed structures in notochordal plates and notochordal sheath are revealed by SRS microscopy with diffraction limited resolution. Our experiment shows that SRS microscopy is an excellent imaging tool for biochemical research with its intrinsic chemical selectivity, high spatiotemporal resolution and native 3D optical sectioning ability.

Lymphatic marker podoplanin/D2-40 in human advanced cirrhotic liver- Re-evaluations of microlymphatic abnormalities

Science.gov (United States)

2010-01-01

Background From the morphological appearance, it was impossible to distinguish terminal portal venules from small lymphatic vessels in the portal tract even using histochemical microscopic techniques. Recently, D2-40 was found to be expressed at a high level in lymphatic endothelial cells (LECs). This study was undertaken to elucidate hepatic lymphatic vessels during progression of cirrhosis by examining the expression of D2-40 in LECs. Methods Surgical wedge biopsy specimens were obtained from non-cirrhotic portions of human livers (normal control) and from cirrhotic livers (LC) (Child A-LC and Child C-LC). Immunohistochemical (IHC), Western blot, and immunoelectron microscopic studies were conducted using D2-40 as markers for lymphatic vessels, as well as CD34 for capillary blood vessels. Results Imunostaining of D2-40 produced a strong reaction in lymphatic vessels only, especially in Child C-LC. It was possible to distinguish the portal venules from the small lymphatic vessels using D-40. Immunoelectron microscopy revealed strong D2-40 expression along the luminal and abluminal portions of the cell membrane of LECs in Child C-LC tissue. Conclusion It is possible to distinguish portal venules from small lymphatic vessels using D2-40 as marker. D2-40- labeling in lymphatic capillary endothelial cells is related to the degree of fibrosis in cirrhotic liver. PMID:21059220

Lymphatic marker podoplanin/D2-40 in human advanced cirrhotic liver- Re-evaluations of microlymphatic abnormalities

Directory of Open Access Journals (Sweden)

Yoshimura Kazunori

2010-11-01

Full Text Available Abstract Background From the morphological appearance, it was impossible to distinguish terminal portal venules from small lymphatic vessels in the portal tract even using histochemical microscopic techniques. Recently, D2-40 was found to be expressed at a high level in lymphatic endothelial cells (LECs. This study was undertaken to elucidate hepatic lymphatic vessels during progression of cirrhosis by examining the expression of D2-40 in LECs. Methods Surgical wedge biopsy specimens were obtained from non-cirrhotic portions of human livers (normal control and from cirrhotic livers (LC (Child A-LC and Child C-LC. Immunohistochemical (IHC, Western blot, and immunoelectron microscopic studies were conducted using D2-40 as markers for lymphatic vessels, as well as CD34 for capillary blood vessels. Results Imunostaining of D2-40 produced a strong reaction in lymphatic vessels only, especially in Child C-LC. It was possible to distinguish the portal venules from the small lymphatic vessels using D-40. Immunoelectron microscopy revealed strong D2-40 expression along the luminal and abluminal portions of the cell membrane of LECs in Child C-LC tissue. Conclusion It is possible to distinguish portal venules from small lymphatic vessels using D2-40 as marker. D2-40- labeling in lymphatic capillary endothelial cells is related to the degree of fibrosis in cirrhotic liver.

Traditional microscopy instruction versus process-oriented virtual microscopy instruction: a naturalistic experiment with control group.

Science.gov (United States)

Helle, Laura; Nivala, Markus; Kronqvist, Pauliina; Gegenfurtner, Andreas; Björk, Pasi; Säljö, Roger

2011-03-30

Virtual microscopy is being introduced in medical education as an approach for learning how to interpret information in microscopic specimens. It is, however, far from evident how to incorporate its use into existing teaching practice. The aim of the study was to explore the consequences of introducing virtual microscopy tasks into an undergraduate pathology course in an attempt to render the instruction more process-oriented. The research questions were: 1) How is virtual microscopy perceived by students? 2) Does work on virtual microscopy tasks contribute to improvement in performance in microscopic pathology in comparison with attending assistant-led demonstrations only? During a one-week period, an experimental group completed three sets of virtual microscopy homework assignments in addition to attending demonstrations. A control group attended the demonstrations only. Performance in microscopic pathology was measured by a pre-test and a post-test. Student perceptions of regular instruction and virtual microscopy were collected one month later by administering the Inventory of Intrinsic Motivation and open-ended questions. The students voiced an appreciation for virtual microscopy for the purposes of the course and for self-study. As for learning gains, the results indicated that learning was speeded up in a subgroup of students consisting of conscientious high achievers. The enriched instruction model may be suited as such for elective courses following the basic course. However, the instructional model needs further development to be suited for basic courses.

Scanning electron microscopy-energy dispersive X-ray spectrometer ...

African Journals Online (AJOL)

The distribution of arsenic (As) and cadmium (Cd) in himematsutake was analyzed using scanning electron microscopy-energy dispersive X-ray spectrometer (SEM-EDX). The atomic percentage of the metals was confirmed by inductively coupled plasma-mass spectrometer (ICP-MS). Results show that the accumulation of ...

The 2015 super-resolution microscopy roadmap

International Nuclear Information System (INIS)

Hell, Stefan W; Sahl, Steffen J; Bates, Mark; Jakobs, Stefan; Zhuang, Xiaowei; Heintzmann, Rainer; Booth, Martin J; Bewersdorf, Joerg; Shtengel, Gleb; Hess, Harald; Tinnefeld, Philip; Honigmann, Alf; Testa, Ilaria; Cognet, Laurent; Lounis, Brahim; Ewers, Helge; Davis, Simon J; Eggeling, Christian; Klenerman, David; Willig, Katrin I

2015-01-01

Far-field optical microscopy using focused light is an important tool in a number of scientific disciplines including chemical, (bio)physical and biomedical research, particularly with respect to the study of living cells and organisms. Unfortunately, the applicability of the optical microscope is limited, since the diffraction of light imposes limitations on the spatial resolution of the image. Consequently the details of, for example, cellular protein distributions, can be visualized only to a certain extent. Fortunately, recent years have witnessed the development of ‘super-resolution’ far-field optical microscopy (nanoscopy) techniques such as stimulated emission depletion (STED), ground state depletion (GSD), reversible saturated optical (fluorescence) transitions (RESOLFT), photoactivation localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), structured illumination microscopy (SIM) or saturated structured illumination microscopy (SSIM), all in one way or another addressing the problem of the limited spatial resolution of far-field optical microscopy. While SIM achieves a two-fold improvement in spatial resolution compared to conventional optical microscopy, STED, RESOLFT, PALM/STORM, or SSIM have all gone beyond, pushing the limits of optical image resolution to the nanometer scale. Consequently, all super-resolution techniques open new avenues of biomedical research. Because the field is so young, the potential capabilities of different super-resolution microscopy approaches have yet to be fully explored, and uncertainties remain when considering the best choice of methodology. Thus, even for experts, the road to the future is sometimes shrouded in mist. The super-resolution optical microscopy roadmap of Journal of Physics D: Applied Physics addresses this need for clarity. It provides guidance to the outstanding questions through a collection of short review articles from experts in the field, giving a thorough

Phase-Modulation Laser Interference Microscopy

DEFF Research Database (Denmark)

Brazhe, Alexey; Brazhe, Nadezda; Maximov, G. V.

2008-01-01

We describe how phase-modulation laser interference microscopy and wavelet analysis can be applied to noninvasive nonstained visualization and study of the structural and dynamical properties of living cells. We show how phase images of erythrocytes can reveal the difference between various...... erythrocyte forms and stages of hemolysis and how phase images of neurons reveal their complex intracellular structure. Temporal variations of the refractive index are analyzed to detect cellular rhythmic activity on different time scales as well as to uncover interactions between the cellular processes....

Labeling Human Mesenchymal Stem Cells with Gold Nanocages for in vitro and in vivo Tracking by Two-Photon Microscopy and Photoacoustic Microscopy

Science.gov (United States)

Zhang, Yu Shrike; Wang, Yu; Wang, Lidai; Wang, Yucai; Cai, Xin; Zhang, Chi; Wang, Lihong V.; Xia, Younan

2013-01-01

Stem cell tracking is a highly important subject. Current techniques based on nanoparticle-labeling, such as magnetic resonance imaging, fluorescence microscopy, and micro-computed tomography, are plagued by limitations including relatively low sensitivity or penetration depth, involvement of ionizing irradiation, and potential cytotoxicity of the nanoparticles. Here we introduce a new class of contrast agents based on gold nanocages (AuNCs) with hollow interiors and porous walls to label human mesenchymal stem cells (hMSCs) for both in vitro and in vivo tracking using two-photon microscopy and photoacoustic microscopy. As demonstrated by the viability assay, the AuNCs showed negligible cytotoxicity under a reasonable dose, and did not alter the differentiation potential of the hMSCs into desired lineages. We were able to image the cells labeled with AuNCs in vitro for at least 28 days in culture, as well as to track the cells that homed to the tumor region in nude mice in vivo. PMID:23946820

Leucine-enkephalin-like immunoreactivity is localized in luteinizing hormone-producing cells in the axolotl (Ambystoma mexicanum) pituitary.

Science.gov (United States)

Suzuki, Hirohumi; Yamamoto, Toshiharu

2014-02-01

In this study, we used immunohistochemical techniques to determine the cell type of leucine-enkephalin (Leu-ENK)-immunoreactive cells in the axolotl (Ambystoma mexicanum) pituitary. Immunoreactive cells were scattered throughout the pars distalis except for the dorso-caudal portion. These cells were immuno-positive for luteinizing hormone (LH), but they were immuno-negative for adrenocorticotrophic, growth, and thyroid-stimulating hormones, as well as prolactin. Immunoelectron microscopy demonstrated that Leu-ENK-like substance and LH co-localized within the same secretory granules. Leu-ENK secreted from gonadotrophs may participate in LH secretion in an autocrine fashion, and/or may participate in the release of sex steroids together with LH. Copyright © 2013 Elsevier Ltd. All rights reserved.

Detailed characterisation of focused ion beam induced lateral damage on silicon carbide samples by electrical scanning probe microscopy and transmission electron microscopy

Science.gov (United States)

Stumpf, F.; Abu Quba, A. A.; Singer, P.; Rumler, M.; Cherkashin, N.; Schamm-Chardon, S.; Cours, R.; Rommel, M.

2018-03-01

The lateral damage induced by focused ion beam on silicon carbide was characterized using electrical scanning probe microscopy (SPM), namely, scanning spreading resistance microscopy and conductive atomic force microscopy (c-AFM). It is shown that the damage exceeds the purposely irradiated circles with a radius of 0.5 μm by several micrometres, up to 8 μm for the maximum applied ion dose of 1018 cm-2. Obtained SPM results are critically compared with earlier findings on silicon. For doses above the amorphization threshold, in both cases, three different areas can be distinguished. The purposely irradiated area exhibits resistances smaller than the non-affected substrate. A second region with strongly increasing resistance and a maximum saturation value surrounds it. The third region shows the transition from maximum resistance to the base resistance of the unaffected substrate. It correlates to the transition from amorphized to defect-rich to pristine crystalline substrate. Additionally, conventional transmission electron microscopy (TEM) and annular dark-field STEM were used to complement and explain the SPM results and get a further understanding of the defect spreading underneath the surface. Those measurements also show three different regions that correlate well with the regions observed from electrical SPM. TEM results further allow to explain observed differences in the electrical results for silicon and silicon carbide which are most prominent for ion doses above 3 × 1016 cm-2. Furthermore, the conventional approach to perform current-voltage measurements by c-AFM was critically reviewed and several improvements for measurement and analysis process were suggested that result in more reliable and impactful c-AFM data.

New tools for comparing microscopy images: quantitative analysis of cell types in Bacillus subtilis.

Science.gov (United States)

van Gestel, Jordi; Vlamakis, Hera; Kolter, Roberto

2015-02-15

Fluorescence microscopy is a method commonly used to examine individual differences between bacterial cells, yet many studies still lack a quantitative analysis of fluorescence microscopy data. Here we introduce some simple tools that microbiologists can use to analyze and compare their microscopy images. We show how image data can be converted to distribution data. These data can be subjected to a cluster analysis that makes it possible to objectively compare microscopy images. The distribution data can further be analyzed using distribution fitting. We illustrate our methods by scrutinizing two independently acquired data sets, each containing microscopy images of a doubly labeled Bacillus subtilis strain. For the first data set, we examined the expression of srfA and tapA, two genes which are expressed in surfactin-producing and matrix-producing cells, respectively. For the second data set, we examined the expression of eps and tapA; these genes are expressed in matrix-producing cells. We show that srfA is expressed by all cells in the population, a finding which contrasts with a previously reported bimodal distribution of srfA expression. In addition, we show that eps and tapA do not always have the same expression profiles, despite being expressed in the same cell type: both operons are expressed in cell chains, while single cells mainly express eps. These findings exemplify that the quantification and comparison of microscopy data can yield insights that otherwise would go unnoticed. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Down-regulation of integrin β1 and focal adhesion kinase in renal glomeruli under various hemodynamic conditions.

Directory of Open Access Journals (Sweden)

Xiaoli Yuan

Full Text Available Given that integrin β1 is an important component of the connection to maintain glomerular structural integrity, by binding with multiple extracellular matrix proteins and mediating intracellular signaling. Focal adhesion kinase (FAK is the most essential intracellular integrator in the integrin β1-FAK signalling pathway. Here, we investigated the changes of the two molecules and visualized the possible interaction between them under various hemodynamic conditions in podocytes. Mice kidney tissues were prepared using in vivo cryotechnique (IVCT and then were stained and observed using light microscopy, confocal laser scanning microscopy and immunoelectron microscopy. The expression of these molecules were examined by western blot. Under the normal condition, integrin β1 stained continually and evenly at the membrane, and FAK was located in the cytoplasm and nuclei of the podocytes. There were significant colocalized plaques of two molecules. But under acute hypertensive and cardiac arrest conditions, integrin β1 decreased and stained intermittently. Similarly, FAK decreased and appeared uneven. Additionally, FAK translocated to the nuclei of the podocytes. As a result, the colocalization of integrin β1 and FAK reduced obviously under these conditions. Western blot assay showed a consistent result with the immunostaining. Collectively, the abnormal redistribution and decreased expressions of integrin β1 and FAK are important molecular events in regulating the functions of podocytes under abnormal hemodynamic conditions. IVCT could offer considerable advantages for morphological analysis when researching renal diseases.

Characterization of human breast cancer by scanning acoustic microscopy

Science.gov (United States)

Chen, Di; Malyarenko, Eugene; Seviaryn, Fedar; Yuan, Ye; Sherman, Mark; Bandyopadhyay, Sudeshna; Gierach, Gretchen; Greenway, Christopher W.; Maeva, Elena; Strumban, Emil; Duric, Neb; Maev, Roman

2013-03-01

Objectives: The purpose of this study was to characterize human breast cancer tissues by the measurement of microacoustic properties. Methods: We investigated eight breast cancer patients using acoustic microscopy. For each patient, seven blocks of tumor tissue were collected from seven different positions around a tumor mass. Frozen sections (10 micrometer, μm) of human breast cancer tissues without staining and fixation were examined in a scanning acoustic microscope with focused transducers at 80 and 200 MHz. Hematoxylin and Eosin (H and E) stained sections from the same frozen breast cancer tissues were imaged by optical microscopy for comparison. Results: The results of acoustic imaging showed that acoustic attenuation and sound speed in cancer cell-rich tissue regions were significantly decreased compared with the surrounding tissue regions, where most components are normal cells/tissues, such as fibroblasts, connective tissue and lymphocytes. Our observation also showed that the ultrasonic properties were influenced by arrangements of cells and tissue patterns. Conclusions: Our data demonstrate that attenuation and sound speed imaging can provide biomechanical information of the tumor and normal tissues. The results also demonstrate the potential of acoustic microscopy as an auxiliary method for operative detection and localization of cancer affected regions.

Scanning electron microscopy of individual nanoparticle bio-markers in liquid

Energy Technology Data Exchange (ETDEWEB)

Liv, Nalan, E-mail: n.liv@tudelft.nl; Lazić, Ivan; Kruit, Pieter; Hoogenboom, Jacob P.

2014-08-01

We investigated SEM imaging of nanoparticle biomarkers suspended below a thin membrane, with the ultimate goal of integrating functional fluorescence and structural SEM measurements of samples kept at ambient or hydrated conditions. In particular, we investigated how resolving power in liquid SEM is affected by the interaction of the electron beam with the membrane. Simulations with the Geant4-based Monte Carlo scheme developed by Kieft and Bosch (2008) [1] are compared to experimental results with suspended nanoparticles. For 20 nm and 50 nm thin membranes, we found a beam broadening of 1.5 nm and 3 nm, respectively, with an excellent agreement between simulations and experiments. 15 nm Au nanoparticles and bio-functionalized core-shell quantum dots can be individually resolved in denser clusters. We demonstrated the imaging of single EGF-conjugated quantum dots docked at filopodia during cellular uptake with both fluorescence microscopy and SEM simultaneously. These results open novel opportunities for correlating live fluorescence microscopy with structural electron microscopy. - Highlights: • We investigate the achievable resolution in liquid scanning electron microscopy (SEM). • We demonstrate liquid SEM imaging of individual fluorescent nanoparticle bio-markers • We show imaging of cellular QDot uptake with simultaneous fluorescence microscopy and SEM. • The positions of individual QDots can be resolved with details on cellular structure.

Scanning electron microscopy of individual nanoparticle bio-markers in liquid

International Nuclear Information System (INIS)

Liv, Nalan; Lazić, Ivan; Kruit, Pieter; Hoogenboom, Jacob P.

2014-01-01

We investigated SEM imaging of nanoparticle biomarkers suspended below a thin membrane, with the ultimate goal of integrating functional fluorescence and structural SEM measurements of samples kept at ambient or hydrated conditions. In particular, we investigated how resolving power in liquid SEM is affected by the interaction of the electron beam with the membrane. Simulations with the Geant4-based Monte Carlo scheme developed by Kieft and Bosch (2008) [1] are compared to experimental results with suspended nanoparticles. For 20 nm and 50 nm thin membranes, we found a beam broadening of 1.5 nm and 3 nm, respectively, with an excellent agreement between simulations and experiments. 15 nm Au nanoparticles and bio-functionalized core-shell quantum dots can be individually resolved in denser clusters. We demonstrated the imaging of single EGF-conjugated quantum dots docked at filopodia during cellular uptake with both fluorescence microscopy and SEM simultaneously. These results open novel opportunities for correlating live fluorescence microscopy with structural electron microscopy. - Highlights: • We investigate the achievable resolution in liquid scanning electron microscopy (SEM). • We demonstrate liquid SEM imaging of individual fluorescent nanoparticle bio-markers • We show imaging of cellular QDot uptake with simultaneous fluorescence microscopy and SEM. • The positions of individual QDots can be resolved with details on cellular structure

Extended Field Laser Confocal Microscopy (EFLCM): Combining automated Gigapixel image capture with in silico virtual microscopy

International Nuclear Information System (INIS)

Flaberg, Emilie; Sabelström, Per; Strandh, Christer; Szekely, Laszlo

2008-01-01

Confocal laser scanning microscopy has revolutionized cell biology. However, the technique has major limitations in speed and sensitivity due to the fact that a single laser beam scans the sample, allowing only a few microseconds signal collection for each pixel. This limitation has been overcome by the introduction of parallel beam illumination techniques in combination with cold CCD camera based image capture. Using the combination of microlens enhanced Nipkow spinning disc confocal illumination together with fully automated image capture and large scale in silico image processing we have developed a system allowing the acquisition, presentation and analysis of maximum resolution confocal panorama images of several Gigapixel size. We call the method Extended Field Laser Confocal Microscopy (EFLCM). We show using the EFLCM technique that it is possible to create a continuous confocal multi-colour mosaic from thousands of individually captured images. EFLCM can digitize and analyze histological slides, sections of entire rodent organ and full size embryos. It can also record hundreds of thousands cultured cells at multiple wavelength in single event or time-lapse fashion on fixed slides, in live cell imaging chambers or microtiter plates. The observer independent image capture of EFLCM allows quantitative measurements of fluorescence intensities and morphological parameters on a large number of cells. EFLCM therefore bridges the gap between the mainly illustrative fluorescence microscopy and purely quantitative flow cytometry. EFLCM can also be used as high content analysis (HCA) instrument for automated screening processes

Measurement of replication structures at the nanometer scale using super-resolution light microscopy.

Science.gov (United States)

Baddeley, D; Chagin, V O; Schermelleh, L; Martin, S; Pombo, A; Carlton, P M; Gahl, A; Domaing, P; Birk, U; Leonhardt, H; Cremer, C; Cardoso, M C

2010-01-01

DNA replication, similar to other cellular processes, occurs within dynamic macromolecular structures. Any comprehensive understanding ultimately requires quantitative data to establish and test models of genome duplication. We used two different super-resolution light microscopy techniques to directly measure and compare the size and numbers of replication foci in mammalian cells. This analysis showed that replication foci vary in size from 210 nm down to 40 nm. Remarkably, spatially modulated illumination (SMI) and 3D-structured illumination microscopy (3D-SIM) both showed an average size of 125 nm that was conserved throughout S-phase and independent of the labeling method, suggesting a basic unit of genome duplication. Interestingly, the improved optical 3D resolution identified 3- to 5-fold more distinct replication foci than previously reported. These results show that optical nanoscopy techniques enable accurate measurements of cellular structures at a level previously achieved only by electron microscopy and highlight the possibility of high-throughput, multispectral 3D analyses.

Transmission electron microscopy, fluorescence microscopy, and confocal raman microscopic analysis of ultrastructural and compositional heterogeneity of Cornus alba L. wood cell wall.

Science.gov (United States)

Ma, Jianfeng; Ji, Zhe; Zhou, Xia; Zhang, Zhiheng; Xu, Feng

2013-02-01

Transmission electron microscopy (TEM), fluorescence microscopy, and confocal Raman microscopy can be used to characterize ultrastructural and compositional heterogeneity of plant cell walls. In this study, TEM observations revealed the ultrastructural characterization of Cornus alba L. fiber, vessel, axial parenchyma, ray parenchyma, and pit membrane between cells, notably with the ray parenchyma consisting of two well-defined layers. Fluorescence microscopy evidenced that cell corner middle lamella was more lignified than adjacent compound middle lamella and secondary wall with variation in lignification level from cell to cell. In situ Raman images showed that the inhomogeneity in cell wall components (cellulose and lignin) among different cells and within morphologically distinct cell wall layers. As the significant precursors of lignin biosynthesis, the pattern of coniferyl alcohol and aldehyde (joint abbreviation Lignin-CAA for both structures) distribution in fiber cell wall was also identified by Raman images, with higher concentration occurring in the fiber secondary wall where there was the highest cellulose concentration. Moreover, noteworthy was the observation that higher concentration of lignin and very minor amounts of cellulose were visualized in the pit membrane areas. These complementary microanalytical methods provide more accurate and complete information with regard to ultrastructural and compositional characterization of plant cell walls.

Confocal scanning microscopy

DEFF Research Database (Denmark)

Bariani, Paolo

This report is based on a metrological investigation on confocal microscopy technique carried out by Uffe Rolf Arlø Theilade and Paolo Bariani. The purpose of the experimental activity was twofold a metrological instrument characterization and application to assessment of rough PP injection moulded...... replicated topography. Confocal microscopy is seen to be a promising technique in metrology of microstructures. Some limitations with respect to surface metrology were found during the experiments. The experiments were carried out using a Zeiss LSM 5 Pascal microscope owned by the Danish Polymer Centre...

A correlative optical microscopy and scanning electron microscopy approach to locating nanoparticles in brain tumors.

Science.gov (United States)

Kempen, Paul J; Kircher, Moritz F; de la Zerda, Adam; Zavaleta, Cristina L; Jokerst, Jesse V; Mellinghoff, Ingo K; Gambhir, Sanjiv S; Sinclair, Robert

2015-01-01

The growing use of nanoparticles in biomedical applications, including cancer diagnosis and treatment, demands the capability to exactly locate them within complex biological systems. In this work a correlative optical and scanning electron microscopy technique was developed to locate and observe multi-modal gold core nanoparticle accumulation in brain tumor models. Entire brain sections from mice containing orthotopic brain tumors injected intravenously with nanoparticles were imaged using both optical microscopy to identify the brain tumor, and scanning electron microscopy to identify the individual nanoparticles. Gold-based nanoparticles were readily identified in the scanning electron microscope using backscattered electron imaging as bright spots against a darker background. This information was then correlated to determine the exact location of the nanoparticles within the brain tissue. The nanoparticles were located only in areas that contained tumor cells, and not in the surrounding healthy brain tissue. This correlative technique provides a powerful method to relate the macro- and micro-scale features visible in light microscopy with the nanoscale features resolvable in scanning electron microscopy. Copyright © 2014 Elsevier Ltd. All rights reserved.

Comparison of Rapid Malaria Test and Laboratory Microscopy ...

African Journals Online (AJOL)

Michael Horsfall

ABSTRACT: Blood samples collected from 272 volunteers in two communities of Bayelsa State in the Niger. Delta area were investigated for falciparum malaria parasite using the rapid test based on the detection of soluble antigen and laboratory microscopy test. The data showed that out of the 272 samples collected, ...

Friction force microscopy study of annealed diamond-like carbon film

International Nuclear Information System (INIS)

Choi, Won Seok; Joung, Yeun-Ho; Heo, Jinhee; Hong, Byungyou

2012-01-01

In this paper we introduce mechanical and structural characteristics of diamond-like carbon (DLC) films which were prepared on silicon substrates by radio frequency (RF) plasma enhanced chemical vapor deposition (PECVD) method using methane (CH 4 ) and hydrogen (H 2 ) gas. The films were annealed at various temperatures ranging from 300 to 900 °C in steps of 200 °C using rapid thermal processor (RTP) in nitrogen ambient. Tribological properties of the DLC films were investigated by atomic force microscopy (AFM) in friction force microscopy (FFM) mode. The structural properties of the films were obtained by high resolution transmission electron microscopy (TEM) and X-ray photoelectron spectroscopy (XPS). The wettability of the films was obtained using contact angle measurement. XPS analysis showed that the sp 3 content is decreased from 75.2% to 24.1% while the sp 2 content is increased from 24.8% to 75.9% when the temperature is changed from 300 to 900 °C. The contact angles of DLC films were higher than 70°. The FFM measurement results show that the highest friction coefficient value was achieved at 900 °C annealing temperature.

Friction force microscopy study of annealed diamond-like carbon film

Energy Technology Data Exchange (ETDEWEB)

Choi, Won Seok; Joung, Yeun-Ho [School of Electrical Engineering, Hanbat National University, Daejeon 305-719 (Korea, Republic of); Heo, Jinhee [Materials Safety Evaluation Group, Korea Institute of Materials Science, Changwon 641-831 (Korea, Republic of); Hong, Byungyou, E-mail: byhong@skku.edu [School of Information and Communication Engineering, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of)

2012-10-15

In this paper we introduce mechanical and structural characteristics of diamond-like carbon (DLC) films which were prepared on silicon substrates by radio frequency (RF) plasma enhanced chemical vapor deposition (PECVD) method using methane (CH{sub 4}) and hydrogen (H{sub 2}) gas. The films were annealed at various temperatures ranging from 300 to 900 °C in steps of 200 °C using rapid thermal processor (RTP) in nitrogen ambient. Tribological properties of the DLC films were investigated by atomic force microscopy (AFM) in friction force microscopy (FFM) mode. The structural properties of the films were obtained by high resolution transmission electron microscopy (TEM) and X-ray photoelectron spectroscopy (XPS). The wettability of the films was obtained using contact angle measurement. XPS analysis showed that the sp{sup 3} content is decreased from 75.2% to 24.1% while the sp{sup 2} content is increased from 24.8% to 75.9% when the temperature is changed from 300 to 900 °C. The contact angles of DLC films were higher than 70°. The FFM measurement results show that the highest friction coefficient value was achieved at 900 °C annealing temperature.

Glucose transporters are expressed in taste receptor cells.

Science.gov (United States)

Merigo, Flavia; Benati, Donatella; Cristofoletti, Mirko; Osculati, Francesco; Sbarbati, Andrea

2011-08-01

In the intestine, changes of sugar concentration generated in the lumen during digestion induce adaptive responses of glucose transporters in the epithelium. A close matching between the intestinal expression of glucose transporters and the composition and amount of the diet has been provided by several experiments. Functional evidence has demonstrated that the regulation of glucose transporters into enterocytes is induced by the sensing of sugar of the enteroendocrine cells through activation of sweet taste receptors (T1R2 and T1R3) and their associated elements of G-protein-linked signaling pathways (e.g. α-gustducin, phospholipase C β type 2 and transient receptor potential channel M5), which are signaling molecules also involved in the perception of sweet substances in the taste receptor cells (TRCs) of the tongue. Considering this phenotypical similarity between the intestinal cells and TRCs, we evaluated whether the TRCs themselves possess proteins of the glucose transport mechanism. Therefore, we investigated the expression of the typical intestinal glucose transporters (i.e. GLUT2, GLUT5 and SGLT1) in rat circumvallate papillae, using immunohistochemistry, double-labeling immunofluorescence, immunoelectron microscopy and reverse transcriptase-polymerase chain reaction analysis. The results showed that GLUT2, GLUT5 and SGLT1 are expressed in TRCs; their immunoreactivity was also observed in cells that displayed staining for α-gustducin and T1R3 receptor. The immunoelectron microscopic results confirmed that GLUT2, GLUT5 and SGLT1 were predominantly expressed in cells with ultrastructural characteristics of chemoreceptor cells. The presence of glucose transporters in TRCs adds a further link between chemosensory information and cellular responses to sweet stimuli that may have important roles in glucose homeostasis, contributing to a better understanding of the pathways implicated in glucose metabolism. © 2011 The Authors. Journal of Anatomy © 2011

B-Spline potential function for maximum a-posteriori image reconstruction in fluorescence microscopy

Directory of Open Access Journals (Sweden)

Shilpa Dilipkumar

2015-03-01

Full Text Available An iterative image reconstruction technique employing B-Spline potential function in a Bayesian framework is proposed for fluorescence microscopy images. B-splines are piecewise polynomials with smooth transition, compact support and are the shortest polynomial splines. Incorporation of the B-spline potential function in the maximum-a-posteriori reconstruction technique resulted in improved contrast, enhanced resolution and substantial background reduction. The proposed technique is validated on simulated data as well as on the images acquired from fluorescence microscopes (widefield, confocal laser scanning fluorescence and super-resolution 4Pi microscopy. A comparative study of the proposed technique with the state-of-art maximum likelihood (ML and maximum-a-posteriori (MAP with quadratic potential function shows its superiority over the others. B-Spline MAP technique can find applications in several imaging modalities of fluorescence microscopy like selective plane illumination microscopy, localization microscopy and STED.

Deep Learning Microscopy

KAUST Repository

Rivenson, Yair

2017-05-12

We demonstrate that a deep neural network can significantly improve optical microscopy, enhancing its spatial resolution over a large field-of-view and depth-of-field. After its training, the only input to this network is an image acquired using a regular optical microscope, without any changes to its design. We blindly tested this deep learning approach using various tissue samples that are imaged with low-resolution and wide-field systems, where the network rapidly outputs an image with remarkably better resolution, matching the performance of higher numerical aperture lenses, also significantly surpassing their limited field-of-view and depth-of-field. These results are transformative for various fields that use microscopy tools, including e.g., life sciences, where optical microscopy is considered as one of the most widely used and deployed techniques. Beyond such applications, our presented approach is broadly applicable to other imaging modalities, also spanning different parts of the electromagnetic spectrum, and can be used to design computational imagers that get better and better as they continue to image specimen and establish new transformations among different modes of imaging.

Actin restructuring during Salmonella typhimurium infection investigated by confocal and super-resolution microscopy

Science.gov (United States)

Han, Jason J.; Kunde, Yuliya A.; Hong-Geller, Elizabeth; Werner, James H.

2014-01-01

We have used super-resolution optical microscopy and confocal microscopy to visualize the cytoskeletal restructuring of HeLa cells that accompanies and enables Salmonella typhimurium internalization. Herein, we report the use of confocal microscopy to verify and explore infection conditions that would be compatible with super-resolution optical microscopy, using Alexa-488 labeled phalloidin to stain the actin cytoskeletal network. While it is well known that actin restructuring and cytoskeletal rearrangements often accompany and assist in bacterial infection, most studies have employed conventional diffraction-limited fluorescence microscopy to explore these changes. Here we show that the superior spatial resolution provided by single-molecule localization methods (such as direct stochastic optical reconstruction microscopy) enables more precise visualization of the nanoscale changes in the actin cytoskeleton that accompany bacterial infection. In particular, we found that a thin (100-nm) ring of actin often surrounds an invading bacteria 10 to 20 min postinfection, with this ring being transitory in nature. We estimate that a few hundred monofilaments of actin surround the S. typhimurium in this heretofore unreported bacterial internalization intermediate.

Automated seeding-based nuclei segmentation in nonlinear optical microscopy.

Science.gov (United States)

Medyukhina, Anna; Meyer, Tobias; Heuke, Sandro; Vogler, Nadine; Dietzek, Benjamin; Popp, Jürgen

2013-10-01

Nonlinear optical (NLO) microscopy based, e.g., on coherent anti-Stokes Raman scattering (CARS) or two-photon-excited fluorescence (TPEF) is a fast label-free imaging technique, with a great potential for biomedical applications. However, NLO microscopy as a diagnostic tool is still in its infancy; there is a lack of robust and durable nuclei segmentation methods capable of accurate image processing in cases of variable image contrast, nuclear density, and type of investigated tissue. Nonetheless, such algorithms specifically adapted to NLO microscopy present one prerequisite for the technology to be routinely used, e.g., in pathology or intraoperatively for surgical guidance. In this paper, we compare the applicability of different seeding and boundary detection methods to NLO microscopic images in order to develop an optimal seeding-based approach capable of accurate segmentation of both TPEF and CARS images. Among different methods, the Laplacian of Gaussian filter showed the best accuracy for the seeding of the image, while a modified seeded watershed segmentation was the most accurate in the task of boundary detection. The resulting combination of these methods followed by the verification of the detected nuclei performs high average sensitivity and specificity when applied to various types of NLO microscopy images.

Photoinduced force microscopy: A technique for hyperspectral nanochemical mapping

Science.gov (United States)

Murdick, Ryan A.; Morrison, William; Nowak, Derek; Albrecht, Thomas R.; Jahng, Junghoon; Park, Sung

2017-08-01

Advances in nanotechnology have intensified the need for tools that can characterize newly synthesized nanomaterials. A variety of techniques has recently been shown which combines atomic force microscopy (AFM) with optical illumination including tip-enhanced Raman spectroscopy (TERS), scattering-type scanning near-field optical microscopy (sSNOM), and photothermal induced resonance microscopy (PTIR). To varying degrees, these existing techniques enable optical spectroscopy with the nanoscale spatial resolution inherent to AFM, thereby providing nanochemical interrogation of a specimen. Here we discuss photoinduced force microscopy (PiFM), a recently developed technique for nanoscale optical spectroscopy that exploits image forces acting between an AFM tip and sample to detect wavelength-dependent polarization within the sample to generate absorption spectra. This approach enables ∼10 nm spatial resolution with spectra that show correlation with macroscopic optical absorption spectra. Unlike other techniques, PiFM achieves this high resolution with virtually no constraints on sample or substrate properties. The applicability of PiFM to a variety of archetypal systems is reported here, highlighting the potential of PiFM as a useful tool for a wide variety of industrial and academic investigations, including semiconducting nanoparticles, nanocellulose, block copolymers, and low dimensional systems, as well as chemical and morphological mixing at interfaces.

High-resolution intravital microscopy.

Directory of Open Access Journals (Sweden)

Volker Andresen

Full Text Available Cellular communication constitutes a fundamental mechanism of life, for instance by permitting transfer of information through synapses in the nervous system and by leading to activation of cells during the course of immune responses. Monitoring cell-cell interactions within living adult organisms is crucial in order to draw conclusions on their behavior with respect to the fate of cells, tissues and organs. Until now, there is no technology available that enables dynamic imaging deep within the tissue of living adult organisms at sub-cellular resolution, i.e. detection at the level of few protein molecules. Here we present a novel approach called multi-beam striped-illumination which applies for the first time the principle and advantages of structured-illumination, spatial modulation of the excitation pattern, to laser-scanning-microscopy. We use this approach in two-photon-microscopy--the most adequate optical deep-tissue imaging-technique. As compared to standard two-photon-microscopy, it achieves significant contrast enhancement and up to 3-fold improved axial resolution (optical sectioning while photobleaching, photodamage and acquisition speed are similar. Its imaging depth is comparable to multifocal two-photon-microscopy and only slightly less than in standard single-beam two-photon-microscopy. Precisely, our studies within mouse lymph nodes demonstrated 216% improved axial and 23% improved lateral resolutions at a depth of 80 µm below the surface. Thus, we are for the first time able to visualize the dynamic interactions between B cells and immune complex deposits on follicular dendritic cells within germinal centers (GCs of live mice. These interactions play a decisive role in the process of clonal selection, leading to affinity maturation of the humoral immune response. This novel high-resolution intravital microscopy method has a huge potential for numerous applications in neurosciences, immunology, cancer research and

High-Resolution Intravital Microscopy

Science.gov (United States)

Andresen, Volker; Pollok, Karolin; Rinnenthal, Jan-Leo; Oehme, Laura; Günther, Robert; Spiecker, Heinrich; Radbruch, Helena; Gerhard, Jenny; Sporbert, Anje; Cseresnyes, Zoltan; Hauser, Anja E.; Niesner, Raluca

2012-01-01

Cellular communication constitutes a fundamental mechanism of life, for instance by permitting transfer of information through synapses in the nervous system and by leading to activation of cells during the course of immune responses. Monitoring cell-cell interactions within living adult organisms is crucial in order to draw conclusions on their behavior with respect to the fate of cells, tissues and organs. Until now, there is no technology available that enables dynamic imaging deep within the tissue of living adult organisms at sub-cellular resolution, i.e. detection at the level of few protein molecules. Here we present a novel approach called multi-beam striped-illumination which applies for the first time the principle and advantages of structured-illumination, spatial modulation of the excitation pattern, to laser-scanning-microscopy. We use this approach in two-photon-microscopy - the most adequate optical deep-tissue imaging-technique. As compared to standard two-photon-microscopy, it achieves significant contrast enhancement and up to 3-fold improved axial resolution (optical sectioning) while photobleaching, photodamage and acquisition speed are similar. Its imaging depth is comparable to multifocal two-photon-microscopy and only slightly less than in standard single-beam two-photon-microscopy. Precisely, our studies within mouse lymph nodes demonstrated 216% improved axial and 23% improved lateral resolutions at a depth of 80 µm below the surface. Thus, we are for the first time able to visualize the dynamic interactions between B cells and immune complex deposits on follicular dendritic cells within germinal centers (GCs) of live mice. These interactions play a decisive role in the process of clonal selection, leading to affinity maturation of the humoral immune response. This novel high-resolution intravital microscopy method has a huge potential for numerous applications in neurosciences, immunology, cancer research and developmental biology

Video-rate resonant scanning multiphoton microscopy

Science.gov (United States)

Kirkpatrick, Nathaniel D.; Chung, Euiheon; Cook, Daniel C.; Han, Xiaoxing; Gruionu, Gabriel; Liao, Shan; Munn, Lance L.; Padera, Timothy P.; Fukumura, Dai; Jain, Rakesh K.

2013-01-01

The abnormal tumor microenvironment fuels tumor progression, metastasis, immune suppression, and treatment resistance. Over last several decades, developments in and applications of intravital microscopy have provided unprecedented insights into the dynamics of the tumor microenvironment. In particular, intravital multiphoton microscopy has revealed the abnormal structure and function of tumor-associated blood and lymphatic vessels, the role of aberrant tumor matrix in drug delivery, invasion and metastasis of tumor cells, the dynamics of immune cell trafficking to and within tumors, and gene expression in tumors. However, traditional multiphoton microscopy suffers from inherently slow imaging rates—only a few frames per second, thus unable to capture more rapid events such as blood flow, lymphatic flow, and cell movement within vessels. Here, we report the development and implementation of a video-rate multiphoton microscope (VR-MPLSM) based on resonant galvanometer mirror scanning that is capable of recording at 30 frames per second and acquiring intravital multispectral images. We show that the design of the system can be readily implemented and is adaptable to various experimental models. As examples, we demonstrate the utility of the system to directly measure flow within tumors, capture metastatic cancer cells moving within the brain vasculature and cells in lymphatic vessels, and image acute responses to changes in a vascular network. VR-MPLSM thus has the potential to further advance intravital imaging and provide new insight into the biology of the tumor microenvironment. PMID:24353926

Microscopy

Science.gov (United States)

Patricia A. Moss; Les Groom

2001-01-01

Microscopy is the study and interpretation of images produced by a microscope. "Interpretation" is the keyword, because the microscope enables one to see structures that are too small or too close together to be resolved by the unaided eye. (The human eye cannot separate two points or lines that are closer together than 0.1 mm.) it is important to...

Analysis of leaf surfaces using scanning ion conductance microscopy.

Science.gov (United States)

Walker, Shaun C; Allen, Stephanie; Bell, Gordon; Roberts, Clive J

2015-05-01

Leaf surfaces are highly complex functional systems with well defined chemistry and structure dictating the barrier and transport properties of the leaf cuticle. It is a significant imaging challenge to analyse the very thin and often complex wax-like leaf cuticle morphology in their natural state. Scanning electron microscopy (SEM) and to a lesser extent Atomic force microscopy are techniques that have been used to study the leaf surface but their remains information that is difficult to obtain via these approaches. SEM is able to produce highly detailed and high-resolution images needed to study leaf structures at the submicron level. It typically operates in a vacuum or low pressure environment and as a consequence is generally unable to deal with the in situ analysis of dynamic surface events at submicron scales. Atomic force microscopy also possess the high-resolution imaging required and can follow dynamic events in ambient and liquid environments, but can over exaggerate small features and cannot image most leaf surfaces due to their inherent roughness at the micron scale. Scanning ion conductance microscopy (SICM), which operates in a liquid environment, provides a potential complementary analytical approach able to address these issues and which is yet to be explored for studying leaf surfaces. Here we illustrate the potential of SICM on various leaf surfaces and compare the data to SEM and atomic force microscopy images on the same samples. In achieving successful imaging we also show that SICM can be used to study the wetting of hydrophobic surfaces in situ. This has potentially wider implications than the study of leaves alone as surface wetting phenomena are important in a range of fundamental and applied studies. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

Force modulation for improved conductive-mode atomic force microscopy

NARCIS (Netherlands)

Koelmans, W.W.; Sebastian, Abu; Despont, Michel; Pozidis, Haris

We present an improved conductive-mode atomic force microscopy (C-AFM) method by modulating the applied loading force on the tip. Unreliable electrical contact and tip wear are the primary challenges for electrical characterization at the nanometer scale. The experiments show that force modulation

Localization of Minodronate in Mouse Femora Through Isotope Microscopy

Science.gov (United States)

Hongo, Hiromi; Sasaki, Muneteru; Kobayashi, Sachio; Hasegawa, Tomoka; Yamamoto, Tomomaya; Tsuboi, Kanako; Tsuchiya, Erika; Nagai, Tomoya; Khadiza, Naznin; Abe, Miki; Kudo, Ai; Oda, Kimimitsu; Henrique Luiz de Freitas, Paulo; Li, Minqi; Yurimoto, Hisayoshi; Amizuka, Norio

2016-01-01

Minodronate is highlighted for its marked and sustained effects on osteoporotic bones. To determine the duration of minodronate’s effects, we have assessed the localization of the drug in mouse bones through isotope microscopy, after labeling it with a stable nitrogen isotope ([15N]-minodronate). In addition, minodronate-treated bones were assessed by histochemistry and transmission electron microscopy (TEM). Eight-week-old male ICR mice received [15N]-minodronate (1 mg/kg) intravenously and were sacrificed after 3 hr, 24 hr, 1 week, and 1 month. Isotope microscopy showed that [15N]-minodronate was present mainly beneath osteoblasts rather than nearby osteoclasts. At 3 hr after minodronate administration, histochemistry and TEM showed osteoclasts with well-developed ruffled borders. However, osteoclasts were roughly attached to the bone surfaces and did not feature ruffled borders at 24 hr after minodronate administration. The numbers of tartrate-resistant acid phosphatase–positive osteoclasts and alkaline phosphatase–reactive osteoblastic area were not reduced suddenly, and apoptotic osteoclasts appeared in 1 week and 1 month after the injections. Von Kossa staining demonstrated that osteoclasts treated with minodronate did not incorporate mineralized bone matrix. Taken together, minodronate accumulates in bone underneath osteoblasts rather than under bone-resorbing osteoclasts; therefore, it is likely that the minodronate-coated bone matrix is resistant to osteoclastic resorption, which results in a long-lasting and bone-preserving effect. PMID:27666429

Volumetry of human taste buds using laser scanning microscopy.

Science.gov (United States)

Just, T; Srur, E; Stachs, O; Pau, H W

2009-10-01

In vivo laser scanning confocal microscopy is a relatively new, non-invasive method for assessment of oral cavity epithelia. The penetration depth of approximately 200-400 microm allows visualisation of fungiform papillae and their taste buds. This paper describes the technique of in vivo volumetry of human taste buds. Confocal laser scanning microscopy used a diode laser at 670 nm for illumination. Digital laser scanning confocal microscopy equipment consisted of the Heidelberg Retina Tomograph HRTII and the Rostock Cornea Module. Volume scans of fungiform papillae were used for three-dimensional reconstruction of the taste bud. This technique supplied information on taste bud structure and enabled measurement and calculation of taste bud volume. Volumetric data from a 23-year-old man over a nine-day period showed only a small deviation in values. After three to four weeks, phenomenological changes in taste bud structures were found (i.e. a significant increase in volume, followed by disappearance of the taste bud and appearance of a new taste bud). The data obtained indicate the potential application of this non-invasive imaging modality: to evaluate variation of taste bud volume in human fungiform papillae with ageing; to study the effects of chorda tympani nerve transection on taste bud volume; and to demonstrate recovery of taste buds in patients with a severed chorda tympani nerve who show recovery of gustatory sensibility after surgery.

Bessel light sheet structured illumination microscopy

Science.gov (United States)

Noshirvani Allahabadi, Golchehr

Biomedical study researchers using animals to model disease and treatment need fast, deep, noninvasive, and inexpensive multi-channel imaging methods. Traditional fluorescence microscopy meets those criteria to an extent. Specifically, two-photon and confocal microscopy, the two most commonly used methods, are limited in penetration depth, cost, resolution, and field of view. In addition, two-photon microscopy has limited ability in multi-channel imaging. Light sheet microscopy, a fast developing 3D fluorescence imaging method, offers attractive advantages over traditional two-photon and confocal microscopy. Light sheet microscopy is much more applicable for in vivo 3D time-lapsed imaging, owing to its selective illumination of tissue layer, superior speed, low light exposure, high penetration depth, and low levels of photobleaching. However, standard light sheet microscopy using Gaussian beam excitation has two main disadvantages: 1) the field of view (FOV) of light sheet microscopy is limited by the depth of focus of the Gaussian beam. 2) Light-sheet images can be degraded by scattering, which limits the penetration of the excitation beam and blurs emission images in deep tissue layers. While two-sided sheet illumination, which doubles the field of view by illuminating the sample from opposite sides, offers a potential solution, the technique adds complexity and cost to the imaging system. We investigate a new technique to address these limitations: Bessel light sheet microscopy in combination with incoherent nonlinear Structured Illumination Microscopy (SIM). Results demonstrate that, at visible wavelengths, Bessel excitation penetrates up to 250 microns deep in the scattering media with single-side illumination. Bessel light sheet microscope achieves confocal level resolution at a lateral resolution of 0.3 micron and an axial resolution of 1 micron. Incoherent nonlinear SIM further reduces the diffused background in Bessel light sheet images, resulting in

Aberrant tropoelastin secretion in MG-63 human osteosarcoma cells

International Nuclear Information System (INIS)

Curtiss, S.W.

1989-01-01

The secretion of newly synthesized tropoelastin, the soluble precursor of the extracellular matrix protein elastin, is not well understood. MG-63 human osteosarcoma cells were found by immunoblot analysis to synthesize 62 kD and 64 kD tropoelastins. Media from 63 cells labelled for five hours with [ 3 H]-valine contain no detectable tropoelastin, unlike media from other tropoelastin-synthesizing cells. Immunoblots of conditioned media and 1Ox-concentrated conditioned media left on the cells for six days also show an absence of tropoelastin from the cell media. No insoluble elastin is associated with the cell layer, as determined by amino acid analysis and electron microscopy of 18-21 day cell cultures. The absence of tropoelastin from the cell medium and elastin from the extracellular matrix indicates that MG63 cells do not secrete tropoelastin as expected, but accumulate it intracellularly. This accumulation is transient: immunoblots and immunofluorescence microscopy show that cells three days after passage have the highest steady-state levels of tropoelastin per cell, that day 8 cells contain lower but still significant amounts of tropoelastin, and that by day 22 tropoelastin is no longer present in the cell cultures. Cell density is a critical factor in the observed pattern of tropoelastin expression. Cells seeded at ten fold their usual initial density have high tropoelastin levels at one day after passage, sooner than cells seeded normally. Tropoelastin also disappears from high density-seeded cells more quickly and is no longer detectable at day 10. Lysosome-like vesicles containing membranous structures appear by immunoelectron microscopy to be the primary site of intracellular tropoelastin localization

Fluorescence Microscopy Gets Faster and Clearer: Roles of Photochemistry and Selective Illumination

Science.gov (United States)

Wolenski, Joseph S.; Julich, Doerthe

2014-01-01

Significant advances in fluorescence microscopy tend be a balance between two competing qualities wherein improvements in resolution and low light detection are typically accompanied by losses in acquisition rate and signal-to-noise, respectively. These trade-offs are becoming less of a barrier to biomedical research as recent advances in optoelectronic microscopy and developments in fluorophore chemistry have enabled scientists to see beyond the diffraction barrier, image deeper into live specimens, and acquire images at unprecedented speed. Selective plane illumination microscopy has provided significant gains in the spatial and temporal acquisition of fluorescence specimens several mm in thickness. With commercial systems now available, this method promises to expand on recent advances in 2-photon deep-tissue imaging with improved speed and reduced photobleaching compared to laser scanning confocal microscopy. Superresolution microscopes are also available in several modalities and can be coupled with selective plane illumination techniques. The combination of methods to increase resolution, acquisition speed, and depth of collection are now being married to common microscope systems, enabling scientists to make significant advances in live cell and in situ imaging in real time. We show that light sheet microscopy provides significant advantages for imaging live zebrafish embryos compared to laser scanning confocal microscopy. PMID:24600334

Optical microscope illumination analysis using through-focus scanning optical microscopy.

Science.gov (United States)

Attota, Ravi Kiran; Park, Haesung

2017-06-15

Misalignment of the aperture diaphragm present in optical microscopes results in angular illumination asymmetry (ANILAS) at the sample plane. Here we show that through-focus propagation of ANILAS results in a lateral image shift with a focus position. This could lead to substantial errors in quantitative results for optical methods that use through-focus images such as three-dimensional nanoparticle tracking, confocal microscopy, and through-focus scanning optical microscopy (TSOM). A correlation exists between ANILAS and the slant in TSOM images. Hence, the slant in the TSOM image can be used to detect, analyze, and rectify the presence of ANILAS.

Bright-field scanning confocal electron microscopy using a double aberration-corrected transmission electron microscope

Energy Technology Data Exchange (ETDEWEB)

Wang, Peng; Behan, Gavin; Kirkland, Angus I. [Department of Materials, University of Oxford, Parks Road, Oxford OX1 3PH (United Kingdom); Nellist, Peter D., E-mail: peter.nellist@materials.ox.ac.uk [Department of Materials, University of Oxford, Parks Road, Oxford OX1 3PH (United Kingdom); Cosgriff, Eireann C.; D' Alfonso, Adrian J.; Morgan, Andrew J.; Allen, Leslie J. [School of Physics, University of Melbourne, Parkville, Victoria 3010 (Australia); Hashimoto, Ayako [Advanced Nano-characterization Center, National Institute for Materials Science (NIMS), 3-13 Sakura, Tsukuba 305-0003 (Japan); Takeguchi, Masaki [Advanced Nano-characterization Center, National Institute for Materials Science (NIMS), 3-13 Sakura, Tsukuba 305-0003 (Japan); High Voltage Electron Microscopy Station, NIMS, 3-13 Sakura, Tsukuba 305-0003 (Japan); Mitsuishi, Kazutaka [Advanced Nano-characterization Center, National Institute for Materials Science (NIMS), 3-13 Sakura, Tsukuba 305-0003 (Japan); Quantum Dot Research Center, NIMS, 3-13 Sakura, Tsukuba 305-0003 (Japan); Shimojo, Masayuki [High Voltage Electron Microscopy Station, NIMS, 3-13 Sakura, Tsukuba 305-0003 (Japan); Advanced Science Research Laboratory, Saitama Institute of Technology, 1690 Fusaiji, Fukaya 369-0293 (Japan)

2011-06-15

Scanning confocal electron microscopy (SCEM) offers a mechanism for three-dimensional imaging of materials, which makes use of the reduced depth of field in an aberration-corrected transmission electron microscope. The simplest configuration of SCEM is the bright-field mode. In this paper we present experimental data and simulations showing the form of bright-field SCEM images. We show that the depth dependence of the three-dimensional image can be explained in terms of two-dimensional images formed in the detector plane. For a crystalline sample, this so-called probe image is shown to be similar to a conventional diffraction pattern. Experimental results and simulations show how the diffracted probes in this image are elongated in thicker crystals and the use of this elongation to estimate sample thickness is explored. -- Research Highlights: {yields} The confocal probe image in a scanning confocal electron microscopy image reveals information about the thickness and height of the crystalline layer. {yields} The form of the contrast in a three-dimensional bright-field scanning confocal electron microscopy image can be explained in terms of the confocal probe image. {yields} Despite the complicated form of the contrast in bright-field scanning confocal electron microscopy, we see that depth information is transferred on a 10 nm scale.

Stochastic Optical Reconstruction Microscopy (STORM).

Science.gov (United States)

Xu, Jianquan; Ma, Hongqiang; Liu, Yang

2017-07-05

Super-resolution (SR) fluorescence microscopy, a class of optical microscopy techniques at a spatial resolution below the diffraction limit, has revolutionized the way we study biology, as recognized by the Nobel Prize in Chemistry in 2014. Stochastic optical reconstruction microscopy (STORM), a widely used SR technique, is based on the principle of single molecule localization. STORM routinely achieves a spatial resolution of 20 to 30 nm, a ten-fold improvement compared to conventional optical microscopy. Among all SR techniques, STORM offers a high spatial resolution with simple optical instrumentation and standard organic fluorescent dyes, but it is also prone to image artifacts and degraded image resolution due to improper sample preparation or imaging conditions. It requires careful optimization of all three aspects-sample preparation, image acquisition, and image reconstruction-to ensure a high-quality STORM image, which will be extensively discussed in this unit. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Electrical characterization of grain boundaries of CZTS thin films using conductive atomic force microscopy techniques

Energy Technology Data Exchange (ETDEWEB)

Muhunthan, N.; Singh, Om Pal [Compound Semiconductor Solar Cell, Physics of Energy Harvesting Division, New Delhi 110012 (India); Toutam, Vijaykumar, E-mail: toutamvk@nplindia.org [Quantum Phenomena and Applications Division, CSIR-National Physical Laboratory, Dr. K.S. Krishnan Marg, New Delhi 110012 (India); Singh, V.N., E-mail: singhvn@nplindia.org [Compound Semiconductor Solar Cell, Physics of Energy Harvesting Division, New Delhi 110012 (India)

2015-10-15

Graphical abstract: Experimental setup for conducting AFM (C-AFM). - Highlights: • Cu{sub 2}ZnSnS{sub 4} (CZTS) thin film was grown by reactive co-sputtering. • The electronic properties were probed using conducting atomic force microscope, scanning Kelvin probe microscopy and scanning capacitance microscopy. • C-AFM current flow mainly through grain boundaries rather than grain interiors. • SKPM indicated higher potential along the GBs compared to grain interiors. • The SCM explains that charge separation takes place at the interface of grain and grain boundary. - Abstract: Electrical characterization of grain boundaries (GB) of Cu-deficient CZTS (Copper Zinc Tin Sulfide) thin films was done using atomic force microscopic (AFM) techniques like Conductive atomic force microscopy (CAFM), Kelvin probe force microscopy (KPFM) and scanning capacitance microscopy (SCM). Absorbance spectroscopy was done for optical band gap calculations and Raman, XRD and EDS for structural and compositional characterization. Hall measurements were done for estimation of carrier mobility. CAFM and KPFM measurements showed that the currents flow mainly through grain boundaries (GB) rather than grain interiors. SCM results showed that charge separation mainly occurs at the interface of grain and grain boundaries and not all along the grain boundaries.

Scanning electron microscopy of superficial white onychomycosis*

Science.gov (United States)

de Almeida Jr., Hiram Larangeira; Boabaid, Roberta Oliveira; Timm, Vitor; Silva, Ricardo Marques e; de Castro, Luis Antonio Suita

2015-01-01

Superficial white onychomycosis is characterized by opaque, friable, whitish superficial spots on the nail plate. We examined an affected halux nail of a 20-year-old male patient with scanning electron microscopy. The mycological examination isolated Trichophyton mentagrophytes. Abundant hyphae with the formation of arthrospores were found on the nail's surface, forming small fungal colonies. These findings showed the great capacity for dissemination of this form of onychomycosis. PMID:26560225

Quantitative imaging of bilirubin by photoacoustic microscopy

Science.gov (United States)

Zhou, Yong; Zhang, Chi; Yao, Da-Kang; Wang, Lihong V.

2013-03-01

Noninvasive detection of both bilirubin concentration and its distribution is important for disease diagnosis. Here we implemented photoacoustic microscopy (PAM) to detect bilirubin distribution. We first demonstrate that our PAM system can measure the absorption spectra of bilirubin and blood. We also image bilirubin distributions in tissuemimicking samples, both without and with blood mixed. Our results show that PAM has the potential to quantitatively image bilirubin in vivo for clinical applications.

Nanoparticle discrimination based on wavelength and lifetime-multiplexed cathodoluminescence microscopy.

Science.gov (United States)

Garming, Mathijs W H; Weppelman, I Gerward C; de Boer, Pascal; Martínez, Felipe Perona; Schirhagl, Romana; Hoogenboom, Jacob P; Moerland, Robert J

2017-08-31

Nanomaterials can be identified in high-resolution electron microscopy images using spectrally-selective cathodoluminescence. Capabilities for multiplex detection can however be limited, e.g., due to spectral overlap or availability of filters. Also, the available photon flux may be limited due to degradation under electron irradiation. Here, we demonstrate single-pass cathodoluminescence-lifetime based discrimination of different nanoparticles, using a pulsed electron beam. We also show that cathodoluminescence lifetime is a robust parameter even when the nanoparticle cathodoluminescence intensity decays over an order of magnitude. We create lifetime maps, where the lifetime of the cathodoluminescence emission is correlated with the emission intensity and secondary-electron images. The consistency of lifetime-based discrimination is verified by also correlating the emission wavelength and the lifetime of nanoparticles. Our results show how cathodoluminescence lifetime provides an additional channel of information in electron microscopy.

Histochemical study of trans-polyisoprene accumulation by spectral confocal laser scanning microscopy and a specific dye showing fluorescence solvatochromism in the rubber-producing plant, Eucommia ulmoides Oliver.

Science.gov (United States)

Nakazawa, Yoshihisa; Takeda, Tsuyoshi; Suzuki, Nobuaki; Hayashi, Tatsushi; Harada, Yoko; Bamba, Takeshi; Kobayashi, Akio

2013-09-01

A microscopic technique combining spectral confocal laser scanning microscopy with a lipophilic fluorescent dye, Nile red, which can emit trans-polyisoprene specific fluorescence, was developed, and unmixed images of synthesized trans-polyisoprene in situ in Eucommia ulmoides were successfully obtained. The images showed that trans-polyisoprene was initially synthesized as granules in non-articulated laticifers that changed shape to fibers during laticifer maturation. Non-articulated laticifers are developed from single laticiferous cells, which are differentiated from surrounding parenchyma cells in the cambium. Therefore, these observations suggested that trans-polyisoprene biosynthesis first started in laticifer cells as granules and then the granules accumulated and fused in the inner space of the laticifers over time. Finally, laticifers were filled with the synthesized trans-polyisoprene, which formed a fibrous structure fitting the laticifers shape. Both trans- and cis-polyisoprene are among the most important polymers naturally produced by plants, and this microscopic technique combined with histological study should provide useful information in the fields of plant histology, bioindustry and phytochemistry.

Optically sectioned in vivo imaging with speckle illumination HiLo microscopy

Science.gov (United States)

Lim, Daryl; Ford, Tim N.; Chu, Kengyeh K.; Mertz, Jerome

2011-01-01

We present a simple wide-field imaging technique, called HiLo microscopy, that is capable of producing optically sectioned images in real time, comparable in quality to confocal laser scanning microscopy. The technique is based on the fusion of two raw images, one acquired with speckle illumination and another with standard uniform illumination. The fusion can be numerically adjusted, using a single parameter, to produce optically sectioned images of varying thicknesses with the same raw data. Direct comparison between our HiLo microscope and a commercial confocal laser scanning microscope is made on the basis of sectioning strength and imaging performance. Specifically, we show that HiLo and confocal 3-D imaging of a GFP-labeled mouse brain hippocampus are comparable in quality. Moreover, HiLo microscopy is capable of faster, near video rate imaging over larger fields of view than attainable with standard confocal microscopes. The goal of this paper is to advertise the simplicity, robustness, and versatility of HiLo microscopy, which we highlight with in vivo imaging of common model organisms including planaria, C. elegans, and zebrafish.

Analysis of enamel microbiopsies in shed primary teeth by Scanning Electron Microscopy (SEM) and Polarizing Microscopy (PM)

International Nuclear Information System (INIS)

Costa de Almeida, Glauce Regina; Molina, Gabriela Ferian; Meschiari, Cesar Arruda; Barbosa de Sousa, Frederico; Gerlach, Raquel Fernanda

2009-01-01

The aims of this study were 1) to verify how close to the theoretically presumed areas are the areas of enamel microbiopsies carried out in vivo or in exfoliated teeth; 2) to test whether the etching solution penetrates beyond the tape borders; 3) to test whether the etching solution demineralizes the enamel in depth. 24 shed upper primary central incisors were randomly divided into two groups: the Rehydrated Teeth Group and the Dry Teeth Group. An enamel microbiopsy was performed, and the enamel microbiopsies were then analyzed by Scanning Electron Microscopy (SEM) and Polarizing Microscopy (PM). Quantitative birefringence measurements were performed. The 'true' etched area was determined by measuring the etched enamel using the NIH Image analysis program. Enamel birefringence was compared using the paired t test. There was a statistically significant difference when the etched areas in the Rehydrated teeth were compared with those of the Dry teeth (p = 0.04). The etched areas varied from - 11.6% to 73.5% of the presumed area in the Rehydrated teeth, and from 6.6% to 61.3% in the Dry teeth. The mean percentage of variation in each group could be used as a correction factor for the etched area. Analysis of PM pictures shows no evidence of in-depth enamel demineralization by the etching solution. No statistically significant differences in enamel birefringence were observed between values underneath and outside the microbiopsy area in the same tooth, showing that no mineral loss occurred below the enamel superficial layer. Our data showed no evidence of in-depth enamel demineralization by the etching solution used in the enamel microbiopsy proposed for primary enamel. This study also showed a variation in the measured diameter of the enamel microbiopsy in nineteen teeth out of twenty four, indicating that in most cases the etching solution penetrated beyond the tape borders.

NICHD Microscopy and Imaging Core (MIC)

Data.gov (United States)

Federal Laboratory Consortium — The NICHD Microscopy and Imaging Core (MIC) is designed as a multi-user research facility providing training and instrumentation for high resolution microscopy and...

Crystals seen on CSF microscopy in a case of suspected subarachnoid haemorrhage

Science.gov (United States)

Weiand, Daniel; Hanning, Ian; Mouhamadou, Moussa; Wearmouth, Debbie

2015-01-01

Although crystals are rarely identified on cerebrospinal fluid (CSF) microscopy, their presence can be of significant diagnostic value. We report a case of oxalate crystals seen on CSF microscopy of a 43-year-old woman. The patient presented with headaches, nausea and vomiting. CT of the head showed a small focus of hyper-density, suspicious of haemorrhage, in the right side of the pontine cistern. CSF cell count was within the normal range. Although no organisms were seen on microscopy, copious oxalate crystals were seen. The same crystals were seen on microscopy of CSF collected in a fluoride oxalate container used for glucose analysis. A follow-up contrast-enhanced CT angiogram did not demonstrate any abnormalities. It transpired that excess CSF had been collected into a fluoride oxalate container. This had subsequently been decanted into a plain container for microbiological analysis. Correct specimen collection should be emphasised when teaching lumbar puncture technique. PMID:26139652

Magnetoelectric force microscopy based on magnetic force microscopy with modulated electric field.

Science.gov (United States)

Geng, Yanan; Wu, Weida

2014-05-01

We present the realization of a mesoscopic imaging technique, namely, the Magnetoelectric Force Microscopy (MeFM), for visualization of local magnetoelectric effect. The basic principle of MeFM is the lock-in detection of local magnetoelectric response, i.e., the electric field-induced magnetization, using magnetic force microscopy. We demonstrate MeFM capability by visualizing magnetoelectric domains on single crystals of multiferroic hexagonal manganites. Results of several control experiments exclude artifacts or extrinsic origins of the MeFM signal. The parameters are tuned to optimize the signal to noise ratio.

Waveguide evanescent field fluorescence microscopy & its application in cell biology

Science.gov (United States)

Hassanzadeh, Abdollah

are captured and collected during the experiment, permitting time lapse analysis. As a proof of concept, we have monitored the response of cells on the waveguide surface to an external lethal agent. Imaging analysis showed very low photobleaching. Therefore photobleaching can be neglected during the experiments. The effects of secondary patterns (inhomogeneities) in the grating and scratches and inhomogeneities in the wave guiding film on the fluorescence background, ultra-thin film and cell-substrate contact regions image were investigated. In conclusion, we developed and established WEFF microscopy and have visualized and quantified solid thin films thicknesses and cell-substrate contact regions. The achieved low scattering results in an improved signal-to-noise ratio and increased sensitivity. Photobleaching and phototoxicity are largely reduced compared to other microscopy techniques. Therefore imaging can be carried out over extended periods and having better temporal resolution without sample damage, such as effect of external agents on the cell-substrate contact regions. Keywords. Waveguide Evanescent Field Fluorescence Microscopy, Evanescent Field, Ion-exchanged Waveguides, TE modes, TM modes, Electromagnetic Field Distribution, Fluorescence, Microscopy, Optical Waveguides, Imaging, Interface, Triton X-100, Focal Contacts, Close Contacts, Cell-Substrate Contact Regions, Cell-Substrate Separation Distances, Photobleaching, Phototoxicity, Cell-Substrate Interaction, Langmuir-Blodgett Films, Phase Separated Lipid Films, SGG 11 Glass, Resolution, Total Internal Reflection, Total Internal Reflection Fluorescence Microscopy, Osteoblast Cells, Grating Coupling, Prism Coupling, Grating.

Transcribed DNA is preferentially located in the perichromatin region of mammalian cell nuclei

Energy Technology Data Exchange (ETDEWEB)

Niedojadlo, Janusz [Centre of Electron Microscopy, University of Lausanne, Bugnon 27, CH-1005 Lausanne (Switzerland); Department of Cell Biology, Institute of General and Molecular Biology, Nicolaus Copernicus University, PL-87-100 Torun (Poland); Perret-Vivancos, Cecile [Centre of Electron Microscopy, University of Lausanne, Bugnon 27, CH-1005 Lausanne (Switzerland); Kalland, Karl-Henning [Centre for Research in Virology, The Gade Institute, University of Bergen, Jonas Liesv. 91, N-5009 Bergen (Norway); Cmarko, Dusan [Centre of Electron Microscopy, University of Lausanne, Bugnon 27, CH-1005 Lausanne (Switzerland); Charles University in Prague, First Faculty of Medicine, Institute of Cellular Biology and Pathology, and Department of Cell Biology, Institute of Physiology, Academy of Sciences of the Czech Republic, v.v.i., Albertov 4, CZ-12801 Prague (Czech Republic); Cremer, Thomas [Department of Biology II, LMU Biozentrum, Grosshaderner Strasse 2, D-82152 Planegg-Martinsried (Germany); Center for Integrated Protein Science Munich (CIPSM), LMU Munich, Munich (Germany); Driel, Roel van, E-mail: r.vandriel@uva.nl [Swammerdam Institute for Life Sciences, University of Amsterdam, P.O. Box 94215, 1090GE Amsterdam (Netherlands); Fakan, Stanislav, E-mail: sfakan@lrz.uni-muenchen.de [Centre of Electron Microscopy, University of Lausanne, Bugnon 27, CH-1005 Lausanne (Switzerland); Department of Biology II, LMU Biozentrum, Grosshaderner Strasse 2, D-82152 Planegg-Martinsried (Germany); Center for Integrated Protein Science Munich (CIPSM), LMU Munich, Munich (Germany)

2011-02-15

The precise localization of transcribed DNA and resulting RNA is an important aspect of the functional architecture of the nucleus. To this end we have developed a novel in situ hybridization approach in combination with immunoelectron microscopy, using sense and anti-sense RNA probes that are derived from total cellular or cytoplasmic poly(A+) RNA. This new technology is much more gentle than classical in situ hybridization using DNA probes and shows excellent preservation of nuclear structure. Carried out on ultrathin sections of fixed and resin-embedded COS-7 cells, it revealed at high resolution the localization of the genes that code for the cellular mRNAs. Quantitative analysis shows that most transcribed DNA is concentrated in the perichromatin region, i.e. the interface between subchromosomal compact chromatin domains and the interchromatin space essentially devoid of DNA. The RNA that is produced is found mainly in the perichromatin region and the interchromatin space. These results imply that in the mammalian nucleus the chromatin fiber is folded so that active genes are predominantly present in the perichromatin region, which is the most prominent site of transcription.

The neuronal Ca(2+) -binding protein 2 (NECAB2) interacts with the adenosine A(2A) receptor and modulates the cell surface expression and function of the receptor.

Science.gov (United States)

Canela, Laia; Luján, Rafael; Lluís, Carme; Burgueño, Javier; Mallol, Josefa; Canela, Enric I; Franco, Rafael; Ciruela, Francisco

2007-09-01

Heptaspanning membrane also known as G protein-coupled receptors (GPCR) do interact with a variety of intracellular proteins whose function is regulate receptor traffic and/or signaling. Using a yeast two-hybrid screen, NECAB2, a neuronal calcium binding protein, was identified as a binding partner for the adenosine A(2A) receptor (A(2A)R) interacting with its C-terminal domain. Co-localization, co-immunoprecipitation and pull-down experiments showed a close and specific interaction between A(2A)R and NECAB2 in both transfected HEK-293 cells and also in rat striatum. Immunoelectron microscopy detection of NECAB2 and A(2A)R in the rat striatopallidal structures indicated that both proteins are co-distributed in the same glutamatergic nerve terminals. The interaction of NECAB2 with A(2A)R modulated the cell surface expression, the ligand-dependent internalization and the receptor-mediated activation of the MAPK pathway. Overall, these results show that A(2A)R interacts with NECAB2 in striatal neurones co-expressing the two proteins and that the interaction is relevant for A(2A)R function.

Electron Microscopy Society of Southern Africa : proceedings

International Nuclear Information System (INIS)

Snyman, H.C.; Coetzee, J.; Coubrough, R.I.

1987-01-01

The proceedings of the 26th annual conference of the Electron Microscopy Society of Southern Africa are presented. Papers were presented on the following topics: techniques and instrumentation used in electron microscopy, and applications of electron microscopy in the life sciences, including applications in medicine, zoology, botany and microbiology. The use of electron microscopy in the physical sciences was also discussed. Separate abstracts were prepared for seven of the papers presented. The remaining papers were considered outside the subject scope of INIS

Diffractive elements performance in chromatic confocal microscopy

International Nuclear Information System (INIS)

Garzon, J; Duque, D; Alean, A; Toledo, M; Meneses, J; Gharbi, T

2011-01-01

The Confocal Laser Scanning Microscopy (CLSM) has been widely used in the semiconductor industry and biomedicine because of its depth discrimination capability. Subsequent to this technique has been developed in recent years Chromatic Confocal Microscopy. This method retains the same principle of confocal and offers the added advantage of removing the axial movement of the moving system. This advantage is usually accomplished with an optical element that generates a longitudinal chromatic aberration and a coding system that relates the axial position of each point of the sample with the wavelength that is focused on each. The present paper shows the performance of compact chromatic confocal microscope when some different diffractive elements are used for generation of longitudinal chromatic aberration. Diffractive elements, according to the process and manufacturing parameters, may have different diffraction efficiency and focus a specific wavelength in a specific focal position. The performance assessment is carried out with various light sources which exhibit an incoherent behaviour and a broad spectral width.

Isotropic differential phase contrast microscopy for quantitative phase bio-imaging.

Science.gov (United States)

Chen, Hsi-Hsun; Lin, Yu-Zi; Luo, Yuan

2018-05-16

Quantitative phase imaging (QPI) has been investigated to retrieve optical phase information of an object and applied to biological microscopy and related medical studies. In recent examples, differential phase contrast (DPC) microscopy can recover phase image of thin sample under multi-axis intensity measurements in wide-field scheme. Unlike conventional DPC, based on theoretical approach under partially coherent condition, we propose a new method to achieve isotropic differential phase contrast (iDPC) with high accuracy and stability for phase recovery in simple and high-speed fashion. The iDPC is simply implemented with a partially coherent microscopy and a programmable thin-film transistor (TFT) shield to digitally modulate structured illumination patterns for QPI. In this article, simulation results show consistency of our theoretical approach for iDPC under partial coherence. In addition, we further demonstrate experiments of quantitative phase images of a standard micro-lens array, as well as label-free live human cell samples. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Scanning probe microscopy experiments in microgravity

International Nuclear Information System (INIS)

Drobek, Tanja; Reiter, Michael; Heckl, Wolfgang M.

2004-01-01

The scanning probe microscopy setups are small, lightweight and do not require vacuum or high voltage supply. In addition, samples can be investigated directly without further preparation. Therefore, these techniques are well-suited for applications in space, in particular, for operation on the International Space Station (ISS) or for high resolution microscopy on planetary missions. A feasibility study for a scanning tunneling microscopy setup was carried out on a parabolic flight campaign in November 2001 in order to test the technical setup for microgravity applications. With a pocket-size design microscope, a graphite surface was imaged under ambient conditions. Atomic resolution was achieved although the quality of the images was inferior in comparison to laboratory conditions. Improvements for future scanning probe microscopy experiments in microgravity are suggested

Chondroitin-6-sulfate-containing proteoglycan: a new component of human skin dermoepidermal junction

DEFF Research Database (Denmark)

Fine, J D; Couchman, J R

1988-01-01

chondroitin sulfate proteoglycan is present in adult, neonatal, and/or fetal skin, and if present, its ultrastructural localization. Indirect immunofluorescence was performed on human adult, neonatal, and fetal skin. To detect the antigen, specimens were pretreated with chondroitinase ABC; absence of enzyme...... treatment served as negative control. Chondroitin sulfate proteoglycan was detectable in linear homogeneous array along the dermoepidermal junction and within vascular (and when present, adnexal) basement membranes in both adult and neonatal skin. In fetal skin, basement membrane staining was noted as early...... as 54 gestational days. Indirect immunoelectron microscopy and NaCl-split skin studies were performed to ultrastructurally localize the antigen; immune deposits were detectable within the lamina densa in chondroitinase-treated skin. These findings demonstrate that chondroitin sulfate proteoglycan...

CD34-positive interstitial cells of the human detrusor

DEFF Research Database (Denmark)

Rasmussen, Helle; Hansen, Alastair; Smedts, Frank

2007-01-01

using a panel of antibodies directed against CD117/c-kit, CD34, CD31, S100, tryptase, neurofilament, NSE, Factor-VIII and GFAP. A striking finding was an interstitial type of cell which is CD34 immunoreactive (CD34-ir) but CD117/c-kit negative. The cells have a tentacular morphology, enveloping...... flattened processes, ramifying primarily in a bipolar fashion. Using immunoelectron microscopy (I-TEM) it was possible to view CD34 gold labelling of cells corresponding to interstitial cells. Although similar CD34-positive cells have been demonstrated in the bowel wall, they have never been described...... in the detrusor. The ontogeny and function of CD34-ir, a kit-negative cell, is unknown, but it may be involved in smooth muscle contraction....

Hyperspectral light sheet microscopy

Science.gov (United States)

Jahr, Wiebke; Schmid, Benjamin; Schmied, Christopher; Fahrbach, Florian O.; Huisken, Jan

2015-09-01

To study the development and interactions of cells and tissues, multiple fluorescent markers need to be imaged efficiently in a single living organism. Instead of acquiring individual colours sequentially with filters, we created a platform based on line-scanning light sheet microscopy to record the entire spectrum for each pixel in a three-dimensional volume. We evaluated data sets with varying spectral sampling and determined the optimal channel width to be around 5 nm. With the help of these data sets, we show that our setup outperforms filter-based approaches with regard to image quality and discrimination of fluorophores. By spectral unmixing we resolved overlapping fluorophores with up to nanometre resolution and removed autofluorescence in zebrafish and fruit fly embryos.

Super-resolution Microscopy in Plant Cell Imaging.

Science.gov (United States)

Komis, George; Šamajová, Olga; Ovečka, Miroslav; Šamaj, Jozef

2015-12-01

Although the development of super-resolution microscopy methods dates back to 1994, relevant applications in plant cell imaging only started to emerge in 2010. Since then, the principal super-resolution methods, including structured-illumination microscopy (SIM), photoactivation localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), and stimulated emission depletion microscopy (STED), have been implemented in plant cell research. However, progress has been limited due to the challenging properties of plant material. Here we summarize the basic principles of existing super-resolution methods and provide examples of applications in plant science. The limitations imposed by the nature of plant material are reviewed and the potential for future applications in plant cell imaging is highlighted. Copyright © 2015 Elsevier Ltd. All rights reserved.

Electronic Blending in Virtual Microscopy

Science.gov (United States)

Maybury, Terrence S.; Farah, Camile S.

2010-01-01

Virtual microscopy (VM) is a relatively new technology that transforms the computer into a microscope. In essence, VM allows for the scanning and transfer of glass slides from light microscopy technology to the digital environment of the computer. This transition is also a function of the change from print knowledge to electronic knowledge, or as…

Light Microscopy at Maximal Precision

Science.gov (United States)

Bierbaum, Matthew; Leahy, Brian D.; Alemi, Alexander A.; Cohen, Itai; Sethna, James P.

2017-10-01

Microscopy is the workhorse of the physical and life sciences, producing crisp images of everything from atoms to cells well beyond the capabilities of the human eye. However, the analysis of these images is frequently little more accurate than manual marking. Here, we revolutionize the analysis of microscopy images, extracting all the useful information theoretically contained in a complex microscope image. Using a generic, methodological approach, we extract the information by fitting experimental images with a detailed optical model of the microscope, a method we call parameter extraction from reconstructing images (PERI). As a proof of principle, we demonstrate this approach with a confocal image of colloidal spheres, improving measurements of particle positions and radii by 10-100 times over current methods and attaining the maximum possible accuracy. With this unprecedented accuracy, we measure nanometer-scale colloidal interactions in dense suspensions solely with light microscopy, a previously impossible feat. Our approach is generic and applicable to imaging methods from brightfield to electron microscopy, where we expect accuracies of 1 nm and 0.1 pm, respectively.

Light Microscopy at Maximal Precision

Directory of Open Access Journals (Sweden)

Matthew Bierbaum

2017-10-01

Full Text Available Microscopy is the workhorse of the physical and life sciences, producing crisp images of everything from atoms to cells well beyond the capabilities of the human eye. However, the analysis of these images is frequently little more accurate than manual marking. Here, we revolutionize the analysis of microscopy images, extracting all the useful information theoretically contained in a complex microscope image. Using a generic, methodological approach, we extract the information by fitting experimental images with a detailed optical model of the microscope, a method we call parameter extraction from reconstructing images (PERI. As a proof of principle, we demonstrate this approach with a confocal image of colloidal spheres, improving measurements of particle positions and radii by 10–100 times over current methods and attaining the maximum possible accuracy. With this unprecedented accuracy, we measure nanometer-scale colloidal interactions in dense suspensions solely with light microscopy, a previously impossible feat. Our approach is generic and applicable to imaging methods from brightfield to electron microscopy, where we expect accuracies of 1 nm and 0.1 pm, respectively.

Microscopy image segmentation tool: Robust image data analysis

Energy Technology Data Exchange (ETDEWEB)

Valmianski, Ilya, E-mail: ivalmian@ucsd.edu; Monton, Carlos; Schuller, Ivan K. [Department of Physics and Center for Advanced Nanoscience, University of California San Diego, 9500 Gilman Drive, La Jolla, California 92093 (United States)

2014-03-15

We present a software package called Microscopy Image Segmentation Tool (MIST). MIST is designed for analysis of microscopy images which contain large collections of small regions of interest (ROIs). Originally developed for analysis of porous anodic alumina scanning electron images, MIST capabilities have been expanded to allow use in a large variety of problems including analysis of biological tissue, inorganic and organic film grain structure, as well as nano- and meso-scopic structures. MIST provides a robust segmentation algorithm for the ROIs, includes many useful analysis capabilities, and is highly flexible allowing incorporation of specialized user developed analysis. We describe the unique advantages MIST has over existing analysis software. In addition, we present a number of diverse applications to scanning electron microscopy, atomic force microscopy, magnetic force microscopy, scanning tunneling microscopy, and fluorescent confocal laser scanning microscopy.

Microscopy image segmentation tool: Robust image data analysis

Science.gov (United States)

Valmianski, Ilya; Monton, Carlos; Schuller, Ivan K.

2014-03-01

We present a software package called Microscopy Image Segmentation Tool (MIST). MIST is designed for analysis of microscopy images which contain large collections of small regions of interest (ROIs). Originally developed for analysis of porous anodic alumina scanning electron images, MIST capabilities have been expanded to allow use in a large variety of problems including analysis of biological tissue, inorganic and organic film grain structure, as well as nano- and meso-scopic structures. MIST provides a robust segmentation algorithm for the ROIs, includes many useful analysis capabilities, and is highly flexible allowing incorporation of specialized user developed analysis. We describe the unique advantages MIST has over existing analysis software. In addition, we present a number of diverse applications to scanning electron microscopy, atomic force microscopy, magnetic force microscopy, scanning tunneling microscopy, and fluorescent confocal laser scanning microscopy.

Microscopy image segmentation tool: Robust image data analysis

International Nuclear Information System (INIS)

Valmianski, Ilya; Monton, Carlos; Schuller, Ivan K.

2014-01-01

We present a software package called Microscopy Image Segmentation Tool (MIST). MIST is designed for analysis of microscopy images which contain large collections of small regions of interest (ROIs). Originally developed for analysis of porous anodic alumina scanning electron images, MIST capabilities have been expanded to allow use in a large variety of problems including analysis of biological tissue, inorganic and organic film grain structure, as well as nano- and meso-scopic structures. MIST provides a robust segmentation algorithm for the ROIs, includes many useful analysis capabilities, and is highly flexible allowing incorporation of specialized user developed analysis. We describe the unique advantages MIST has over existing analysis software. In addition, we present a number of diverse applications to scanning electron microscopy, atomic force microscopy, magnetic force microscopy, scanning tunneling microscopy, and fluorescent confocal laser scanning microscopy

Confocal microscopy as an early relapse marker for acanthamoeba keratitis.

Science.gov (United States)

Daas, Loay; Viestenz, Arne; Schnabel, Philipp Albert; Fries, Fabian N; Hager, Tobias; SzentmÁry, Nora; Seitz, Berthold

2018-01-01

Acanthameoba keratitis is a serious ophthalmological condition with a potentially vision-threatening prognosis. Early diagnosis and recognition of relapse, and the detection of persistent Acanthamoeba cysts, are essential for informing the prognosis and managing the condition. We suggest the use of in vivo confocal microscopy not only to identify the early signs of relapse after keratoplasty in patients with Acanthamoeba keratitis, but also as an additional follow-up tool after antimicrobial crosslinking. This study shows that in vivo confocal microscopy is, in experienced hands, a quick and reliable diagnostic tool. Clin. Anat. 31:60-63, 2018. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Transmission-type angle deviation microscopy

International Nuclear Information System (INIS)

Chiu, M.-H.; Lai, C.-W.; Tan, C.-T.; Lai, C.-F.

2008-01-01

We present a new microscopy technique that we call transmission angle deviation microscopy (TADM). It is based on common-path heterodyne interferometry and geometrical optics. An ultrahigh sensitivity surface plasmon resonance (SPR) angular sensor is used to expand dynamic measurement ranges and to improve the axial resolution in three-dimensional optical microscopy. When transmitted light is incident upon a specimen, the beam converges or diverges because of refractive and/or surface height variations. Advantages include high axial resolution (∼32 nm), nondestructive and noncontact measurement, and larger measurement ranges (± 80 μm) for a numerical aperture of 0.21in a transparent measurement medium. The technique can be used without conductivity and pretreatment

Resolution enhancement in integral microscopy by physical interpolation.

Science.gov (United States)

Llavador, Anabel; Sánchez-Ortiga, Emilio; Barreiro, Juan Carlos; Saavedra, Genaro; Martínez-Corral, Manuel

2015-08-01

Integral-imaging technology has demonstrated its capability for computing depth images from the microimages recorded after a single shot. This capability has been shown in macroscopic imaging and also in microscopy. Despite the possibility of refocusing different planes from one snap-shot is crucial for the study of some biological processes, the main drawback in integral imaging is the substantial reduction of the spatial resolution. In this contribution we report a technique, which permits to increase the two-dimensional spatial resolution of the computed depth images in integral microscopy by a factor of √2. This is made by a double-shot approach, carried out by means of a rotating glass plate, which shifts the microimages in the sensor plane. We experimentally validate the resolution enhancement as well as we show the benefit of applying the technique to biological specimens.

Adenoviral protein V promotes a process of viral assembly through nucleophosmin 1

Energy Technology Data Exchange (ETDEWEB)

Ugai, Hideyo; Dobbins, George C.; Wang, Minghui [Division of Human Gene Therapy, Departments of Medicine, Obstetrics and Gynecology, Pathology, and Surgery, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Le, Long P. [Massachusetts General Hospital, Pathology Service, 55 Fruit St.-GRJ 249, Boston, MA 02114 (United States); Matthews, David A. [School of Cellular and Molecular Medicine, Medical Sciences Building, University of Bristol, Bristol BS8 1TD (United Kingdom); Curiel, David T., E-mail: dcuriel@radonc.wustl.edu [Division of Human Gene Therapy, Departments of Medicine, Obstetrics and Gynecology, Pathology, and Surgery, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); The Gene Therapy Center, University of Alabama at Birmingham, Birmingham, AL 35294 (United States)

2012-10-25

Adenoviral infection induces nucleoplasmic redistribution of a nucleolar nucleophosmin 1/NPM1/B23.1. NPM1 is preferentially localized in the nucleoli of normal cells, whereas it is also present at the nuclear matrix in cancer cells. However, the biological roles of NPM1 during infection are unknown. Here, by analyzing a pV-deletion mutant, Ad5-dV/TSB, we demonstrate that pV promotes the NPM1 translocation from the nucleoli to the nucleoplasm in normal cells, and the NPM1 translocation is correlated with adenoviral replication. Lack of pV causes a dramatic reduction of adenoviral replication in normal cells, but not cancer cells, and Ad5-dV/TSB was defective in viral assembly in normal cells. NPM1 knockdown inhibits adenoviral replication, suggesting an involvement of NPM1 in adenoviral biology. Further, we show that NPM1 interacts with empty adenovirus particles which are an intermediate during virion maturation by immunoelectron microscopy. Collectively, these data implicate that pV participates in a process of viral assembly through NPM1.

The association of metabotropic glutamate receptor type 5 with the neuronal Ca2+-binding protein 2 modulates receptor function.

Science.gov (United States)

Canela, Laia; Fernández-Dueñas, Víctor; Albergaria, Catarina; Watanabe, Masahiko; Lluís, Carme; Mallol, Josefa; Canela, Enric I; Franco, Rafael; Luján, Rafael; Ciruela, Francisco

2009-10-01

Metabotropic glutamate (mGlu) receptors mediate in part the CNS effects of glutamate. These receptors interact with a large array of intracellular proteins in which the final role is to regulate receptor function. Here, using co-immunoprecipitation and pull-down experiments we showed a close and specific interaction between mGlu(5) receptor and NECAB2 in both transfected human embryonic kidney cells and rat hippocampus. Interestingly, in pull-down experiments increasing concentrations of calcium drastically reduced the ability of these two proteins to interact, suggesting that NECAB2 binds to mGlu(5) receptor in a calcium-regulated manner. Immunoelectron microscopy detection of NECAB2 and mGlu(5) receptor in the rat hippocampal formation indicated that both proteins are codistributed in the same subcellular compartment of pyramidal cells. In addition, the NECAB2/mGlu(5) receptor interaction regulated mGlu(5b)-mediated activation of both inositol phosphate accumulation and the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway. Overall, these findings indicate that NECAB2 by its physical interaction with mGlu(5b) receptor modulates receptor function.

Nonstructural protein 5A is incorporated into hepatitis C virus low-density particle through interaction with core protein and microtubules during intracellular transport.

Directory of Open Access Journals (Sweden)

Chao-Kuen Lai

Full Text Available Nonstructural protein 5A (NS5A of hepatitis C virus (HCV serves dual functions in viral RNA replication and virus assembly. Here, we demonstrate that HCV replication complex along with NS5A and Core protein was transported to the lipid droplet (LD through microtubules, and NS5A-Core complexes were then transported from LD through early-to-late endosomes to the plasma membrane via microtubules. Further studies by cofractionation analysis and immunoelectron microscopy of the released particles showed that NS5A-Core complexes, but not NS4B, were present in the low-density fractions, but not in the high-density fractions, of the HCV RNA-containing virions and associated with the internal virion core. Furthermore, exosomal markers CD63 and CD81 were also detected in the low-density fractions, but not in the high-density fractions. Overall, our results suggest that HCV NS5A is associated with the core of the low-density virus particles which exit the cell through a preexisting endosome/exosome pathway and may contribute to HCV natural infection.

Correlative Super-Resolution Microscopy: New Dimensions and New Opportunities.

Science.gov (United States)

Hauser, Meghan; Wojcik, Michal; Kim, Doory; Mahmoudi, Morteza; Li, Wan; Xu, Ke

2017-06-14

Correlative microscopy, the integration of two or more microscopy techniques performed on the same sample, produces results that emphasize the strengths of each technique while offsetting their individual weaknesses. Light microscopy has historically been a central method in correlative microscopy due to its widespread availability, compatibility with hydrated and live biological samples, and excellent molecular specificity through fluorescence labeling. However, conventional light microscopy can only achieve a resolution of ∼300 nm, undercutting its advantages in correlations with higher-resolution methods. The rise of super-resolution microscopy (SRM) over the past decade has drastically improved the resolution of light microscopy to ∼10 nm, thus creating exciting new opportunities and challenges for correlative microscopy. Here we review how these challenges are addressed to effectively correlate SRM with other microscopy techniques, including light microscopy, electron microscopy, cryomicroscopy, atomic force microscopy, and various forms of spectroscopy. Though we emphasize biological studies, we also discuss the application of correlative SRM to materials characterization and single-molecule reactions. Finally, we point out current limitations and discuss possible future improvements and advances. We thus demonstrate how a correlative approach adds new dimensions of information and provides new opportunities in the fast-growing field of SRM.

Structured illumination microscopy and its new developments

Directory of Open Access Journals (Sweden)

Jianling Chen

2016-05-01

Full Text Available Optical microscopy allows us to observe the biological structures and processes within living cells. However, the spatial resolution of the optical microscopy is limited to about half of the wavelength by the light diffraction. Structured illumination microscopy (SIM, a type of new emerging super-resolution microscopy, doubles the spatial resolution by illuminating the specimen with a patterned light, and the sample and light source requirements of SIM are not as strict as the other super-resolution microscopy. In addition, SIM is easier to combine with the other imaging techniques to improve their imaging resolution, leading to the developments of diverse types of SIM. SIM has great potential to meet the various requirements of living cells imaging. Here, we review the recent developments of SIM and its combination with other imaging techniques.

Super-resolved linear fluorescence localization microscopy using photostable fluorophores: A virtual microscopy study

Science.gov (United States)

Birk, Udo; Szczurek, Aleksander; Cremer, Christoph

2017-12-01

Current approaches to overcome the conventional limit of the resolution potential of light microscopy (of about 200 nm for visible light), often suffer from non-linear effects, which render the quantification of the image intensities in the reconstructions difficult, and also affect the quantification of the biological structure under investigation. As an attempt to face these difficulties, we discuss a particular method of localization microscopy which is based on photostable fluorescent dyes. The proposed method can potentially be implemented as a fast alternative for quantitative localization microscopy, circumventing the need for the acquisition of thousands of image frames and complex, highly dye-specific imaging buffers. Although the need for calibration remains in order to extract quantitative data (such as the number of emitters), multispectral approaches are largely facilitated due to the much less stringent requirements on imaging buffers. Furthermore, multispectral acquisitions can be readily obtained using commercial instrumentation such as e.g. the conventional confocal laser scanning microscope.

Changes in fine structure of pericytes and novel desmin-immunopositive perivascular cells during postnatal development in rat anterior pituitary gland.

Science.gov (United States)

Jindatip, Depicha; Fujiwara, Ken; Horiguchi, Kotaro; Tsukada, Takehiro; Kouki, Tom; Yashiro, Takashi

2013-09-01

Pericytes are perivascular cells associated with capillaries. We previously demonstrated that pericytes, identified by desmin immunohistochemistry, produce type I and III collagens in the anterior pituitary gland of adult rats. In addition, we recently used desmin immunoelectron microscopy to characterize a novel type of perivascular cell, dubbed a desmin-immunopositive perivascular cell, in the anterior pituitary. These two types of perivascular cells differ in fine structure. The present study attempted to characterize the morphological features of pituitary pericytes and novel desmin-immunopositive perivascular cells during postnatal development, in particular their role in collagen synthesis. Desmin immunostaining revealed numerous perivascular cells at postnatal day 5 (P5) and P10. Transmission electron microscopy showed differences in the fine structure of the two cell types, starting at P5. Pericytes had well-developed rough endoplasmic reticulum and Golgi apparatus at P5 and P10. The novel desmin-immunopositive perivascular cells exhibited dilated cisternae of rough endoplasmic reticulum at P5-P30. In addition, during early postnatal development in the gland, a number of type I and III collagen-expressing cells were observed, as were high expression levels of these collagen mRNAs. We conclude that pituitary pericytes and novel desmin-immunopositive perivascular cells contain well-developed cell organelles and that they actively synthesize collagens during the early postnatal period.

Imaging of phase change materials below a capping layer using correlative infrared near-field microscopy and electron microscopy

Science.gov (United States)

Lewin, M.; Hauer, B.; Bornhöfft, M.; Jung, L.; Benke, J.; Michel, A.-K. U.; Mayer, J.; Wuttig, M.; Taubner, T.

2015-10-01

Phase Change Materials (PCM) show two stable states in the solid phase with significantly different optical and electronic properties. They can be switched reversibly between those two states and are promising candidates for future non-volatile memory applications. The development of phase change devices demands characterization tools, yielding information about the switching process at high spatial resolution. Scattering-type Scanning Near-field Optical Microscopy (s-SNOM) allows for spectroscopic analyses of the different optical properties of the PCMs on the nm-scale. By correlating the optical s-SNOM images with transmission electron microscopy images of the same sample, we unambiguously demonstrate the correlation of the infrared optical contrast with the structural state of the phase change material. The investigated sample consists of sandwiched amorphous and crystalline regions of Ag 4 In 3 Sb 67 Te 26 below a 100 nm thick ( ZnS ) 80 - ( SiO2 ) 20 capping layer. Our results demonstrate the sensitivity of s-SNOM to small dielectric near-field contrasts even below a comparably thick capping layer ( 100 nm ).

Leakage radiation interference microscopy.

Science.gov (United States)

Descrovi, Emiliano; Barakat, Elsie; Angelini, Angelo; Munzert, Peter; De Leo, Natascia; Boarino, Luca; Giorgis, Fabrizio; Herzig, Hans Peter

2013-09-01

We present a proof of principle for a new imaging technique combining leakage radiation microscopy with high-resolution interference microscopy. By using oil immersion optics it is demonstrated that amplitude and phase can be retrieved from optical fields, which are evanescent in air. This technique is illustratively applied for mapping a surface mode propagating onto a planar dielectric multilayer on a thin glass substrate. The surface mode propagation constant estimated after Fourier transformation of the measured complex field is well matched with an independent measurement based on back focal plane imaging.

Uncertainties of size measurements in electron microscopy characterization of nanomaterials in foods

DEFF Research Database (Denmark)

Dudkiewicz, Agnieszka; Boxall, Alistair B. A.; Chaudhry, Qasim

2015-01-01

Electron microscopy is a recognized standard tool for nanomaterial characterization, and recommended by the European Food Safety Authority for the size measurement of nanomaterials in food. Despite this, little data have been published assessing the reliability of the method, especially for size...... measurement of nanomaterials characterized by a broad size distribution and/or added to food matrices. This study is a thorough investigation of the measurement uncertainty when applying electron microscopy for size measurement of engineered nanomaterials in foods. Our results show that the number of measured...

4Pi-confocal microscopy of live cells

Science.gov (United States)

Bahlmann, Karsten; Jakobs, Stefan; Hell, Stefan W.

2002-06-01

By coherently adding the spherical wavefronts of two opposing lenses, two-photon excitation 4Pi-confocal fluorescence microscopy has achieved three-dimensional imaging with an axial resolution 3-7 times better than confocal microscopy. So far this improvement was possible only in glycerol-mounted, fixed cells. Here we report 4Pi-confocal microscopy of watery objects and its application to the imaging of live cells. Water immersion 4Pi-confocal microscopy of membrane stained live Escherichia coli bacteria attains a 4.3 fold better axial resolution as compared to the best water immersion confocal microscope. The resolution enhancement results into a vastly improved three-dimensional representation of the bacteria. The first images of live biological samples with an all-directional resolution in the 190-280 nm range are presented here, thus establishing a new resolution benchmark in live cell microscopy.

Two-photon Microscopy and Polarimetry for Assessment of Myocardial Tissue Organization

Science.gov (United States)

Archambault-Wallenburg, Marika

Optical methods can provide useful tissue characterization tools. For this project, two-photon microscopy and polarized light examinations (polarimetry) were used to assess the organizational state of myocardium in healthy, infarcted, and stem-cell regenerated states. Two-photon microscopy visualizes collagen through second-harmonic generation and myocytes through two-photon excitation autofluorescence, providing information on the composition and structure/organization of the tissue. Polarimetry measurements yield a value of linear retardance that can serve as an indicator of tissue anisotropy, and with a dual-projection method, information about the anisotropy axis orientation can also be extracted. Two-photon microscopy results reveal that stem-cell treated tissue retains more myocytes and structure than infarcted myocardium, while polarimetry findings suggest that the injury caused by temporary ligation of a coronary artery is less severe and more diffuse that than caused by a permanent ligation. Both these methods show potential for tissue characterization.

High-speed atomic force microscopy combined with inverted optical microscopy for studying cellular events.

OpenAIRE

Suzuki, Yuki; Sakai, Nobuaki; Yoshida, Aiko; Uekusa, Yoshitsugu; Yagi, Akira; Imaoka, Yuka; Ito, Shuichi; Karaki, Koichi; Takeyasu, Kunio

2013-01-01

A hybrid atomic force microscopy (AFM)-optical fluorescence microscopy is a powerful tool for investigating cellular morphologies and events. However, the slow data acquisition rates of the conventional AFM unit of the hybrid system limit the visualization of structural changes during cellular events. Therefore, high-speed AFM units equipped with an optical/fluorescence detection device have been a long-standing wish. Here we describe the implementation of high-speed AFM coupled with an optic...

Super-resolution microscopy reveals functional organization of dopamine transporters into cholesterol and neuronal activity-dependent nanodomains

DEFF Research Database (Denmark)

Rahbek-Clemmensen, Troels; Lycas, Matthew D.; Erlendsson, Simon

2017-01-01

is dynamically sequestrated into cholesterol-dependent nanodomains in the plasma membrane of presynaptic varicosities and neuronal projections of dopaminergic neurons. Stochastic optical reconstruction microscopy reveals irregular dopamine transporter nanodomains (∼70 nm mean diameter) that were highly sensitive...... to cholesterol depletion. Live photoactivated localization microscopy shows a similar dopamine transporter membrane organization in live heterologous cells. In neurons, dual-color dSTORM shows that tyrosine hydroxylase and vesicular monoamine transporter-2 are distinctively localized adjacent to...

High-resolution MR imaging of triangular fibrocartilage complex (TFCC): comparison of microscopy coils and a conventional small surface coil

Energy Technology Data Exchange (ETDEWEB)

Yoshioka, Hiroshi [Department of Radiology, University of Tsukuba, Tsukuba (Japan); Department of Radiology, Brigham and Women' s Hospital, 75 Francis Street, 02115, Boston, MA (United States); Ueno, Teruko; Itai, Yuji [Department of Radiology, University of Tsukuba, Tsukuba (Japan); Tanaka, Toshikazu [Department of Orthopedic Surgery, Tsukuba Kinen Hospital, Tsukuba (Japan); Shindo, Masashi [Tsukuba University Hospital, Tsukuba (Japan)

2003-10-01

To compare MR images of the triangular fibrocartilage complex (TFCC) using microscopy coils with those using a conventional surface coil qualitatively and quantitatively. Proton density-weighted images and T2*-weighted images of the TFCC from ten normal volunteers were obtained with a conventional surface coil (C4 coil; 80 mm in diameter), a 47-mm microscopy surface coil and a 23-mm microscopy surface coil at 1.5 T. Qualitative image analysis of MR images with three coils was performed by two radiologists who assigned one of five numerical scores (0, nonvisualization; 1, poor; 2, average; 3, good; 4, excellent) for five TFCC components, which were disc proper, triangular ligament, meniscus homologue, ulnotriquetral and ulnolunate ligament. Quantitative analysis included the signal-to-noise ratio (S/N) of the disc proper of TFCC, the lunate cartilage, the lunate bone and the contrast-noise-ratio (C/N) between articular cartilage and disc proper or bone marrow were measured. All structures show higher scores qualitatively on MR with microscopy coils than those with a C4 coil, and the difference was significant with the exception of the ulnolunate ligament. MR with microscopy coils showed significantly higher S/N values than those with a conventional surface coil (P<0.05 to P<0.001). T2*-weighted images using microscopy coils showed significantly higher cartilage-disc proper C/N and cartilage-bone marrow C/N (P<0.01 to P<0.001). On proton density-weighted images, the C/N between cartilage and disc proper with two microscopy coils was significantly higher (P<0.01) than that with a conventional coil. High-resolution MR images of the normal wrist using microscopy coils were superior to those using a conventional surface coil qualitatively and quantitatively. High-resolution MR imaging with a microscopy coil would be a promising method to diagnose TFCC lesions. (orig.)

Assessment of fibrotic liver disease with multimodal nonlinear optical microscopy

Science.gov (United States)

Lu, Fake; Zheng, Wei; Tai, Dean C. S.; Lin, Jian; Yu, Hanry; Huang, Zhiwei

2010-02-01

Liver fibrosis is the excessive accumulation of extracellular matrix proteins such as collagens, which may result in cirrhosis, liver failure, and portal hypertension. In this study, we apply a multimodal nonlinear optical microscopy platform developed to investigate the fibrotic liver diseases in rat models established by performing bile duct ligation (BDL) surgery. The three nonlinear microscopy imaging modalities are implemented on the same sectioned tissues of diseased model sequentially: i.e., second harmonic generation (SHG) imaging quantifies the contents of the collagens, the two-photon excitation fluorescence (TPEF) imaging reveals the morphology of hepatic cells, while coherent anti-Stokes Raman scattering (CARS) imaging maps the distributions of fats or lipids quantitatively across the tissue. Our imaging results show that during the development of liver fibrosis (collagens) in BDL model, fatty liver disease also occurs. The aggregated concentrations of collagen and fat constituents in liver fibrosis model show a certain correlationship between each other.

MULTISCALE TENSOR ANISOTROPIC FILTERING OF FLUORESCENCE MICROSCOPY FOR DENOISING MICROVASCULATURE.

Science.gov (United States)

Prasath, V B S; Pelapur, R; Glinskii, O V; Glinsky, V V; Huxley, V H; Palaniappan, K

2015-04-01

Fluorescence microscopy images are contaminated by noise and improving image quality without blurring vascular structures by filtering is an important step in automatic image analysis. The application of interest here is to automatically extract the structural components of the microvascular system with accuracy from images acquired by fluorescence microscopy. A robust denoising process is necessary in order to extract accurate vascular morphology information. For this purpose, we propose a multiscale tensor with anisotropic diffusion model which progressively and adaptively updates the amount of smoothing while preserving vessel boundaries accurately. Based on a coherency enhancing flow with planar confidence measure and fused 3D structure information, our method integrates multiple scales for microvasculature preservation and noise removal membrane structures. Experimental results on simulated synthetic images and epifluorescence images show the advantage of our improvement over other related diffusion filters. We further show that the proposed multiscale integration approach improves denoising accuracy of different tensor diffusion methods to obtain better microvasculature segmentation.

Macroscopy and Microscopy Study of Scopraia dulcis L.

Directory of Open Access Journals (Sweden)

Djoko Santosa

2018-04-01

Full Text Available A morphological diversity analysis of Scoparia dulcis by macroscopy and microscopy has been conducted. The aim of this research is to determine the morphological and anatomical character of S. dulcis which grow in Opak watershed, Special Region of Yogyakarta. Samples were taken from Sleman (Cangkringan, Ngemplak Kalasan, and Prambanan and Bantul (Imogiri and Pundong region. Selection of sampling location is based on altitude difference. The samples are the third leaf order from the tip of the stem. They were measured in length and width. Microscopy analysis was performed by observing the number of glandular trichomes, palisade cells in the epidermis and stomata in the upper and lower epidermis. The data obtained were analyzed using principal component analysis (PCA. The results show that based on morphological and anatomical assessment, there were found 3 clusters of S. dulcis, i.e. Cangkringan-Ngemplak-Prambanan, Kalasan, and Imogiri-Pundong cluster.

Vibrational Imaging with High Sensitivity via Epidetected Coherent Anti-Stokes Raman Scattering Microscopy

International Nuclear Information System (INIS)

Volkmer, Andreas; Cheng, Ji-Xin; Sunney Xie, X.

2001-01-01

We demonstrate theoretically and experimentally a novel epidetection scheme for coherent anti-Stokes Raman scattering (CARS) microscopy that significantly improves the detection sensitivity. Calculations show that epidetected CARS (E-CARS) signals are present for scatterers smaller than the wavelength of light, whereas the large background signals from the surrounding bulk solvent are suppressed by destructive interference. E-CARS microscopy is capable of revealing small intracellular features that are otherwise buried by the strong water CARS signal

Localisation of N-acetylaspartate in oligodendrocytes/myelin.

Science.gov (United States)

Nordengen, Kaja; Heuser, Christoph; Rinholm, Johanne Egge; Matalon, Reuben; Gundersen, Vidar

2015-03-01

The role of N-acetylaspartate in the brain is unclear. Here we used specific antibodies against N-acetylaspartate and immunocytochemistry of carbodiimide-fixed adult rodent brain to show that, besides staining of neuronal cell bodies in the grey matter, N-acetylaspartate labelling was present in oligodendrocytes/myelin in white matter tracts. Immunoelectron microscopy of the rat hippocampus showed that N-acetylaspartate was concentrated in the myelin. Also neuronal cell bodies and axons contained significant amounts of N-acetylaspartate, while synaptic elements and astrocytes were low in N-acetylaspartate. Mitochondria in axons and neuronal cell bodies contained higher levels of N-acetylaspartate compared to the cytosol, compatible with synthesis of N-acetylaspartate in mitochondria. In aspartoacylase knockout mice, in which catabolism of N-acetylaspartate is blocked, the levels of N-acetylaspartate were largely increased in oligodendrocytes/myelin. In these mice, the highest myelin concentration of N-acetylaspartate was found in the cerebellum, a region showing overt dysmyelination. In organotypic cortical slice cultures there was no evidence for N-acetylaspartate-induced myelin toxicity, supporting the notion that myelin damage is induced by the lack of N-acetylaspartate for lipid production. Our findings also implicate that N-acetylaspartate signals on magnetic resonance spectroscopy reflect not only vital neurons but also vital oligodendrocytes/myelin.

Imaging transient blood vessel fusion events in zebrafish by correlative volume electron microscopy.

Directory of Open Access Journals (Sweden)

Hannah E J Armer

Full Text Available The study of biological processes has become increasingly reliant on obtaining high-resolution spatial and temporal data through imaging techniques. As researchers demand molecular resolution of cellular events in the context of whole organisms, correlation of non-invasive live-organism imaging with electron microscopy in complex three-dimensional samples becomes critical. The developing blood vessels of vertebrates form a highly complex network which cannot be imaged at high resolution using traditional methods. Here we show that the point of fusion between growing blood vessels of transgenic zebrafish, identified in live confocal microscopy, can subsequently be traced through the structure of the organism using Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM and Serial Block Face/Scanning Electron Microscopy (SBF/SEM. The resulting data give unprecedented microanatomical detail of the zebrafish and, for the first time, allow visualization of the ultrastructure of a time-limited biological event within the context of a whole organism.

MRI with microscopy coil of the proximal interphalangeal joints: preliminary study

International Nuclear Information System (INIS)

Liu Min; Sun Haixing; Liu Dimin; Hu Meiyu; Pan Shunping; Wang Ping; Huang Xiaoling; Men Quanfei; Chen Yingming

2009-01-01

Objective: To investigate the use of a microscopy coil in MR examination of proximal interphalangeal joints (PIJ) to collect evidence on micro-anatomical pathological changes for further MR diagnosis. Methods: Four PIJ in cadaver and 12 PIJ from 7 normal volunteers were scanned with a microscopy surface coil (23 mm in diameter) at 1.5 T MRI. Sagittal T 1 -weighted images were also obtained with a conventional surface coil using the same parameters for the volunteers. Based on the observation of sagittal, traverse, coronal PIJ imaging features of cadaver, the optimized sequences were chosen for the volunteer's application. Image quality of the PIJ structures from volunteers was analyzed by two radiologists on a 5-point scale (0, nonvisualization; 1, poor; 2, average; 3, good; 4, excellent) and compared with Wilcoxon signed tank test between the microscopy and C3 surface coil in sagittal direction. Results: With microscopy coil all sequences can visualize the main structures of PI J, and all the PIJ images were classified as good or excellent except for one as average, while all the 12 PIJ with C3 coil were classified as poor or invisible. PIJ structures (proximal phalanx head, middle phalanx base, cartilage, volar plate) from volunteers show higher scores of quality with microscopy coils than those with C3 coil, and the difference was significant (Z =-3.274, -3.274, -3.213, and -3.742 respectively, all P<0.01). Conclusion: High-resolution MRI of the normal PIJ with microscopy coil was superior to those with a conventional surface coil, and it can be a promising method to diagnose interphalangeal joints lesions. (authors)

Sputum Microscopy With Fluorescein Diacetate Predicts Tuberculosis Infectiousness.

Science.gov (United States)

Datta, Sumona; Sherman, Jonathan M; Tovar, Marco A; Bravard, Marjory A; Valencia, Teresa; Montoya, Rosario; Quino, Willi; D'Arcy, Nikki; Ramos, Eric S; Gilman, Robert H; Evans, Carlton A

2017-09-01

Sputum from patients with tuberculosis contains subpopulations of metabolically active and inactive Mycobacterium tuberculosis with unknown implications for infectiousness. We assessed sputum microscopy with fluorescein diacetate (FDA, evaluating M. tuberculosis metabolic activity) for predicting infectiousness. Mycobacterium tuberculosis was quantified in pretreatment sputum of patients with pulmonary tuberculosis using FDA microscopy, culture, and acid-fast microscopy. These 35 patients' 209 household contacts were followed with prevalence surveys for tuberculosis disease for 6 years. FDA microscopy was positive for a median of 119 (interquartile range [IQR], 47-386) bacteria/µL sputum, which was 5.1% (IQR, 2.4%-11%) the concentration of acid-fast microscopy-positive bacteria (2069 [IQR, 1358-3734] bacteria/μL). Tuberculosis was diagnosed during follow-up in 6.4% (13/209) of contacts. For patients with lower than median concentration of FDA microscopy-positive M. tuberculosis, 10% of their contacts developed tuberculosis. This was significantly more than 2.7% of the contacts of patients with higher than median FDA microscopy results (crude hazard ratio [HR], 3.8; P = .03). This association maintained statistical significance after adjusting for disease severity, chemoprophylaxis, drug resistance, and social determinants (adjusted HR, 3.9; P = .02). Mycobacterium tuberculosis that was FDA microscopy negative was paradoxically associated with greater infectiousness. FDA microscopy-negative bacteria in these pretreatment samples may be a nonstaining, slowly metabolizing phenotype better adapted to airborne transmission. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America.

Immunoelectron microscopic characterization of nucleolus-associated domains during hibernation.

Science.gov (United States)

Malatesta, Manuela; Zancanaro, Carlo; Biggiogera, Marco

2011-01-01

The nucleolus represents a highly dynamic nuclear compartment involved in multiple functions and able to promptly respond to variations of metabolic needs. In the hibernator dormouse, which drastically modifies its metabolic activity during the seasonal cycle, the nucleolus undergoes structural and molecular changes during the torpor bouts; in particular, it shows many nucleoplasmic invaginations containing weakly contrasted areas of unknown nature. To analyze the molecular composition of these nucleolus-associated domains (NADs) and to understand their functional significance, the fine nucleolar composition has been investigated by means of ultrastructural immunocytochemistry in different tissues of euthermic, hibernating, and arousing hazel dormice (Muscardinus avellanarius): in particular, the intranucleolar location of several protein factors involved in the transcription and processing of either pre-rRNA or pre-mRNA has been considered. NADs proved to form during hibernation and disappear upon arousal and were found to contain m₃-G-capped snRNAs, snRNPs, hnRNPs, and the survival motor neuron protein; they were, on the contrary, devoid of the nucleolar factors tested (polymerase I, fibrillarin, nucleolin, and the ribosomal phosphoproteins P₀, P₁, and P₂). We hypothesize that NADs may represent a transient storage site for those molecules involved in the pre-mRNA splicing, which usually transit through the nucleolus; upon arousal, this would facilitate the resumption of RNA maturation by promoting the rapid reactivation of the molecular trafficking from the nucleolus. © 2010 Wiley-Liss, Inc.

Application of super-resolution optical microscopy in biology

International Nuclear Information System (INIS)

Mao Xiuhai; Du Jiancong; Huang Qing; Fan Chunhai; Deng Suhui

2013-01-01

Background: A noninvasive, real-time far-field optical microscopy is needed to study the dynamic function inside cells and proteins. However, the resolution limit of traditional optical microscope is about 200 nm due to the diffraction limit of light. So, it's hard to directly observe the subcellular structures. Over the past several years of microscopy development, the diffraction limit of fluorescence microscopy has been overcome and its resolution limit is about tens of nanometers. Methods: To overcome the diffraction limit of light, many super-resolution fluoresce microscopes, including stimulated emission of depletion microscopy (STED), photoactivation localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), have been developed. Conclusions: These methods have been applied in cell biology, microbiology and neurobiology, and the technology of super-resolution provides a new insight into the life science. (authors)

Scanning probe microscopy in material science and biology

International Nuclear Information System (INIS)

Cricenti, A; Colonna, S; Girasole, M; Gori, P; Ronci, F; Longo, G; Dinarelli, S; Luce, M; Rinaldi, M; Ortenzi, M

2011-01-01

A review of the activity of scanning probe microscopy at our Institute is presented, going from instrumentation to software development of scanning tunnelling microscopy, atomic force microscopy and scanning near-field optical microscopy (SNOM). Some of the most important experiments in material science and biology performed by our group through the years with these SPM techniques will be presented. Finally, infrared applications by coupling a SNOM with a free electron laser will also be presented.

Automated analysis of high-content microscopy data with deep learning.

Science.gov (United States)

Kraus, Oren Z; Grys, Ben T; Ba, Jimmy; Chong, Yolanda; Frey, Brendan J; Boone, Charles; Andrews, Brenda J

2017-04-18

Existing computational pipelines for quantitative analysis of high-content microscopy data rely on traditional machine learning approaches that fail to accurately classify more than a single dataset without substantial tuning and training, requiring extensive analysis. Here, we demonstrate that the application of deep learning to biological image data can overcome the pitfalls associated with conventional machine learning classifiers. Using a deep convolutional neural network (DeepLoc) to analyze yeast cell images, we show improved performance over traditional approaches in the automated classification of protein subcellular localization. We also demonstrate the ability of DeepLoc to classify highly divergent image sets, including images of pheromone-arrested cells with abnormal cellular morphology, as well as images generated in different genetic backgrounds and in different laboratories. We offer an open-source implementation that enables updating DeepLoc on new microscopy datasets. This study highlights deep learning as an important tool for the expedited analysis of high-content microscopy data. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

Microscopy of the hair and trichogram

Directory of Open Access Journals (Sweden)

Özlem Dicle

2014-06-01

Full Text Available Hair microscopy is a fast and simple method for the diagnosis of various disorders affecting the hair in daily practice. For the microscopy of the hair, samples are collected by either clipping or plucking. The trichogram technique which the hair sample is collected by a standardized plucking method is used for the diagnosis of hair shedding and of alopecia via hair root pattern. In this review, the examination techniques and details are discussed and the most common indications for the hair microscopy including hair abnormalities as a part of genodermatosis and, infections and infestations affecting the hair are highlighted.

Rapid diagnosis of malaria by fluorescent microscopy with light microscope and interface filter

International Nuclear Information System (INIS)

Hussain, I.; Tayyib, M.; Farooq, M.; Ahmed, N.

2008-01-01

The present study is planned to compare acridine orange (A.O) staining with Giemsa staining by using light microscopy with IF and also with fluorescent microscopy for detection of parasites in peripheral blood of patients suffering from clinically suspected cases of malaria. 200 patients with fever and shivering were included. General investigations like Hb, TLC and platelets were done by sysmex K-1000. Thin and thick blood films were made and stained according to protocol given i.e. by Giemsa and AO stains and slides were examined by different microscopes i.e. light microscope, light microscope with IFS and fluorescent microscope. Out of 200 subjects, 170 (85%) patients showed positive parasitaemia and 30 (15%) subjects were negative for malaria parasites. fib, TLC and platelets were reduced when comparing with MP negative cases. IFS microscope with acridine orange staining showed early detection of malaria parasites by counting fewer fields as compared to light microscopy with Giemsa stains. Time consumed for detection of parasites was also significantly reduced in IFS microscope by using AO stains. (author)

Comparison of confocal microscopy and two-photon microscopy in mouse cornea in vivo.

Science.gov (United States)

Lee, Jun Ho; Lee, Seunghun; Gho, Yong Song; Song, In Seok; Tchah, Hungwon; Kim, Myoung Joon; Kim, Ki Hean

2015-03-01

High-resolution imaging of the cornea is important for studying corneal diseases at cellular levels. Confocal microscopy (CM) has been widely used in the clinic, and two-photon microscopy (TPM) has recently been introduced in various pre-clinical studies. We compared the performance of CM and TPM in normal mouse corneas and neovascularized mouse corneas induced by suturing. Balb/C mice and C57BL/6 mice expressing green fluorescent protein (GFP) were used to compare modalities based on intrinsic contrast and extrinsic fluorescence contrast. CM based on reflection (CMR), CM based on fluorescence (CMF), and TPM based on intrinsic/extrinsic fluorescence and second harmonic generation (SHG) were compared by imaging the same sections of mouse corneas sequentially in vivo. In normal mouse corneas, CMR visualized corneal cell morphologies with some background noise, and CMF visualized GFP expressing corneal cells clearly. TPM visualized corneal cells and collagen in the stroma based on fluorescence and SHG, respectively. However, in neovascularized mouse corneas, CMR could not resolve cells deep inside the cornea due to high background noise from the effects of increased structural irregularity induced by suturing. CMF and TPM visualized cells and induced vasculature better than CMR because both collect signals from fluorescent cells only. Both CMF and TPM had signal decays with depth due to the structural irregularity, with CMF having faster signal decay than TPM. CMR, CMF, and TPM showed different degrees of image degradation in neovascularized mouse corneas. Copyright © 2015 Elsevier Ltd. All rights reserved.

Facile method to stain the bacterial cell surface for super-resolution fluorescence microscopy

Energy Technology Data Exchange (ETDEWEB)

Gunsolus, Ian L.; Hu, Dehong; Mihai, Cosmin; Lohse, Samuel E.; Lee, Chang-Soo; Torelli, Marco; Hamers, Robert J.; Murphy, Catherine; Orr, Galya; Haynes, Christy L.

2014-01-01

A method to fluorescently stain the surfaces of both Gram-negative and Gram-positive bacterial cells compatible with super-resolution fluorescence microscopy is presented. This method utilizes a commercially-available fluorescent probe to label primary amines at the surface of the cell. We demonstrate efficient staining of two bacterial strains, the Gram-negative Shewanella oneidensis MR-1 and the Gram-positive Bacillus subtilis 168. Using structured illumination microscopy and stochastic optical reconstruction microscopy, which require high quantum yield or specialized dyes, we show that this staining method may be used to resolve the bacterial cell surface with sub-diffraction-limited resolution. We further use this method to identify localization patterns of nanomaterials, specifically cadmium selenide quantum dots, following interaction with bacterial cells.

Growth of Pd-Filled Carbon Nanotubes on the Tip of Scanning Probe Microscopy

Directory of Open Access Journals (Sweden)

Tomokazu Sakamoto

2009-01-01

Full Text Available We have synthesized Pd-filled carbon nanotubes (CNTs oriented perpendicular to Si substrates using a microwave plasma-enhanced chemical vapor deposition (MPECVD for the application of scanning probe microscopy (SPM tip. Prior to the CVD growth, Al thin film (10 nm was coated on the substrate as a buffer layer followed by depositing a 5∼40 nm-thick Pd film as a catalyst. The diameter and areal density of CNTs grown depend largely on the initial Pd thickness. Scanning electron microscopy (SEM and transmission electron microscopy (TEM images clearly show that Pd is successfully encapsulated into the CNTs, probably leading to higher conductivity. Using optimum growth conditions, Pd-filled CNTs are successfully grown on the apex of the conventional SPM cantilever.

Atomic resolution scanning tunneling microscopy in a cryogen free dilution refrigerator at 15 mK

International Nuclear Information System (INIS)

Haan, A. M. J. den; Wijts, G. H. C. J.; Galli, F.; Oosterkamp, T. H.; Usenko, O.; Baarle, G. J. C. van; Zalm, D. J. van der

2014-01-01

Pulse tube refrigerators are becoming more common, because they are cost efficient and demand less handling than conventional (wet) refrigerators. However, a downside of a pulse tube system is the vibration level at the cold-head, which is in most designs several micrometers. We implemented vibration isolation techniques which significantly reduced vibration levels at the experiment. These optimizations were necessary for the vibration sensitive magnetic resonance force microscopy experiments at milli-kelvin temperatures for which the cryostat is intended. With these modifications we show atomic resolution scanning tunneling microscopy on graphite. This is promising for scanning probe microscopy applications at very low temperatures

Three axis vector magnet set-up for cryogenic scanning probe microscopy

International Nuclear Information System (INIS)

Galvis, J. A.; Herrera, E.; Buendía, A.; Guillamón, I.; Vieira, S.; Suderow, H.; Azpeitia, J.; Luccas, R. F.; Munuera, C.; García-Hernandez, M.

2015-01-01

We describe a three axis vector magnet system for cryogenic scanning probe microscopy measurements. We discuss the magnet support system and the power supply, consisting of a compact three way 100 A current source. We obtain tilted magnetic fields in all directions with maximum value of 5T along z-axis and of 1.2T for XY-plane magnetic fields. We describe a scanning tunneling microscopy-spectroscopy (STM-STS) set-up, operating in a dilution refrigerator, which includes a new high voltage ultralow noise piezodrive electronics and discuss the noise level due to vibrations. STM images and STS maps show atomic resolution and the tilted vortex lattice at 150 mK in the superconductor β-Bi 2 Pd. We observe a strongly elongated hexagonal lattice, which corresponds to the projection of the tilted hexagonal vortex lattice on the surface. We also discuss Magnetic Force Microscopy images in a variable temperature insert

Three axis vector magnet set-up for cryogenic scanning probe microscopy

Energy Technology Data Exchange (ETDEWEB)

Galvis, J. A. [Laboratorio de Bajas Temperaturas, Departamento de Física de la Materia Condensada, Instituto de Ciencia de Materiales Nicolás Cabrera, Condensed Matter Physics Center (IFIMAC), Facultad de Ciencias Universidad Autónoma de Madrid, 28049 Madrid (Spain); Departamento de Ciencias Naturales Facultad de Ingeniería Universidad Central, Bogotá (Colombia); Herrera, E.; Buendía, A. [Laboratorio de Bajas Temperaturas, Departamento de Física de la Materia Condensada, Instituto de Ciencia de Materiales Nicolás Cabrera, Condensed Matter Physics Center (IFIMAC), Facultad de Ciencias Universidad Autónoma de Madrid, 28049 Madrid (Spain); Guillamón, I.; Vieira, S.; Suderow, H. [Laboratorio de Bajas Temperaturas, Departamento de Física de la Materia Condensada, Instituto de Ciencia de Materiales Nicolás Cabrera, Condensed Matter Physics Center (IFIMAC), Facultad de Ciencias Universidad Autónoma de Madrid, 28049 Madrid (Spain); Unidad Asociada de Bajas Temperaturas y Altos Campos Magnéticos, UAM, CSIC, Cantoblanco, E-28049 Madrid (Spain); Azpeitia, J.; Luccas, R. F.; Munuera, C.; García-Hernandez, M. [Unidad Asociada de Bajas Temperaturas y Altos Campos Magnéticos, UAM, CSIC, Cantoblanco, E-28049 Madrid (Spain); Instituto de Ciencia de Materiales de Madrid, Consejo Superior de Investigaciones Científicas (ICMM-CSIC), Sor Juana Inés de la Cruz 3, 28049 Madrid (Spain); and others

2015-01-15

We describe a three axis vector magnet system for cryogenic scanning probe microscopy measurements. We discuss the magnet support system and the power supply, consisting of a compact three way 100 A current source. We obtain tilted magnetic fields in all directions with maximum value of 5T along z-axis and of 1.2T for XY-plane magnetic fields. We describe a scanning tunneling microscopy-spectroscopy (STM-STS) set-up, operating in a dilution refrigerator, which includes a new high voltage ultralow noise piezodrive electronics and discuss the noise level due to vibrations. STM images and STS maps show atomic resolution and the tilted vortex lattice at 150 mK in the superconductor β-Bi{sub 2}Pd. We observe a strongly elongated hexagonal lattice, which corresponds to the projection of the tilted hexagonal vortex lattice on the surface. We also discuss Magnetic Force Microscopy images in a variable temperature insert.

From Nano to Macro: Studying the Hierarchical Structure of the Corneal Extracellular Matrix

Science.gov (United States)

Quantock, Andrew J.; Winkler, Moritz; Parfitt, Geraint J.; Young, Robert D.; Brown, Donald J.; Boote, Craig; Jester, James V.

2014-01-01

In this review, we discuss current methods for studying ocular extracellular matrix (ECM) assembly from the ‘nano’ to the ‘macro’ levels of hierarchical organization. Since collagen is the major structural protein in the eye, providing mechanical strength and controlling ocular shape, the methods presented focus on understanding the molecular assembly of collagen at the nanometer level using x-ray scattering through to the millimeter to centimeter level using nonlinear optical (NLO) imaging of second harmonic generated (SHG) signals. Three-dimensional analysis of ECM structure is also discussed, including electron tomography, serial block face scanning electron microscopy (SBF-SEM) and digital image reconstruction. Techniques to detect non-collagenous structural components of the ECM are also presented, and these include immunoelectron microscopy and staining with cationic dyes. Together, these various approaches are providing new insights into the structural blueprint of the ocular ECM, and in particular that of the cornea, which impacts upon our current understanding of the control of corneal shape, pathogenic mechanisms underlying ectatic disorders of the cornea and the potential for corneal tissue engineering. PMID:25819457

The cyclic nucleotide gated cation channel AtCNGC10 traffics from the ER via Golgi vesicles to the plasma membrane of Arabidopsis root and leaf cells

Directory of Open Access Journals (Sweden)

Andres Marilou A

2007-09-01

Full Text Available Abstract Background The cyclic nucleotide-gated ion channels (CNGCs maintain cation homeostasis essential for a wide range of physiological processes in plant cells. However, the precise subcellular locations and trafficking of these membrane proteins are poorly understood. This is further complicated by a general deficiency of information about targeting pathways of membrane proteins in plants. To investigate CNGC trafficking and localization, we have measured Atcngc5 and Atcngc10 expression in roots and leaves, analyzed AtCNGC10-GFP fusions transiently expressed in protoplasts, and conducted immunofluorescence labeling of protoplasts and immunoelectron microscopic analysis of high pressure frozen leaves and roots. Results AtCNGC10 mRNA and protein levels were 2.5-fold higher in roots than leaves, while AtCNGC5 mRNA and protein levels were nearly equal in these tissues. The AtCNGC10-EGFP fusion was targeted to the plasma membrane in leaf protoplasts, and lightly labeled several intracellular structures. Immunofluorescence microscopy with affinity purified CNGC-specific antisera indicated that AtCNGC5 and AtCNGC10 are present in the plasma membrane of protoplasts. Immunoelectron microscopy demonstrated that AtCNGC10 was associated with the plasma membrane of mesophyll, palisade parenchyma and epidermal cells of leaves, and the meristem, columella and cap cells of roots. AtCNCG10 was also observed in the endoplasmic reticulum and Golgi cisternae and vesicles of 50–150 nm in size. Patch clamp assays of an AtCNGC10-GFP fusion expressed in HEK293 cells measured significant cation currents. Conclusion AtCNGC5 and AtCNGC10 are plasma membrane proteins. We postulate that AtCNGC10 traffics from the endoplasmic reticulum via the Golgi apparatus and associated vesicles to the plasma membrane. The presence of the cation channel, AtCNGC10, in root cap meristem cells, cell plate, and gravity-sensing columella cells, combined with the previously reported

Advanced Electron Microscopy in Materials Physics

International Nuclear Information System (INIS)

Zhu, Y.; Jarausch, K.

2009-01-01

Aberration correction has opened a new frontier in electron microscopy by overcoming the limitations of conventional round lenses, providing sub-angstrom-sized probes and extending information limits. The imaging and analytical performance of these corrector-equipped microscopes affords an unprecedented opportunity to study structure-property relationships of matter at the atomic scale. This new generation of microscopes is able to retrieve high-quality structural information comparable to neutron and synchrotron x-ray experiments, but with local atomic resolution. These advances in instrumentation are accelerating the research and development of various functional materials ranging from those for energy generation, conversion, transportation and storage to those for catalysis and nano-device applications. The dramatic improvements in electron-beam illumination and detection also present a host of new challenges for the interpretation and optimization of experiments. During 7-9 November 2007, a workshop, entitled 'Aberration Corrected Electron Microscopy in Material Physics', was convened at the Center for Functional Nanomaterials, Brookhaven National Laboratories (BNL) to address these opportunities and challenges. The workshop was co-sponsored by Hitachi High Technologies, a leader in electron microscopy instrumentation, and BNL's Institute of Advanced Electron Microscopy, a leader in materials physics research using electron microscopy. The workshop featured presentations by internationally prominent scientists working at the frontiers of electron microscopy, both on developing instrumentation and applying it in materials physics. The meeting, structured to stimulate scientific exchanges and explore new capabilities, brought together ∼100 people from over 10 countries. This special issue complies many of the advances in instrument performance and materials physics reported by the invited speakers and attendees at the workshop.

A review of cellphone microscopy for disease detection.

Science.gov (United States)

Dendere, R; Myburg, N; Douglas, T S

2015-12-01

The expansion in global cellphone network coverage coupled with advances in cellphone imaging capabilities present an opportunity for the advancement of cellphone microscopy as a low-cost alternative to conventional microscopy for disease detection in resource-limited regions. The development of cellphone microscopy has also benefitted from the availability of low-cost miniature microscope components such as low-power light-emitting diodes and ball lenses. As a result, researchers are developing hardware and software techniques that would enable such microscopes to produce high-resolution, diagnostic-quality images. This approach may lead to more widespread delivery of diagnostic services in resource-limited areas where there is a shortage of the skilled labour required for conventional microscopy and where prevalence of infectious and other diseases is still high. In this paper, we review current techniques, clinical applications and challenges faced in the field of cellphone microscopy. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

Mueller matrix microscopy on a Morpho butterfly

International Nuclear Information System (INIS)

Arteaga, Oriol; Kuntman, Ertan; Antó, Joan; Pascual, Esther; Canillas, Adolf; Bertran, Enric

2015-01-01

The brilliant iridescent colouring in male Morpho butterflies is due to the microstrutures and nanostructures present in the wing scales, rather than pigments. In this work Mueller matrix microscopy is used to investigate the polarization properties of butterfly wing scales in reflection and transmission. It is found that the top layer of more transparent scales (cover scales) have very different polarimetric properties from the ground iridescent scales. Images with high spatial resolution showing the retarding and diattenuating optical properties for both types of scales are provided. (paper)

Ultrasonically synthesized organic liquid-filled chitosan microcapsules: part 2: characterization using AFM (atomic force microscopy) and combined AFM-confocal laser scanning fluorescence microscopy.

Science.gov (United States)

Mettu, Srinivas; Ye, Qianyu; Zhou, Meifang; Dagastine, Raymond; Ashokkumar, Muthupandian

2018-04-25

Atomic Force Microscopy (AFM) is used to measure the stiffness and Young's modulus of individual microcapsules that have a chitosan cross-linked shell encapsulating tetradecane. The oil filled microcapsules were prepared using a one pot synthesis via ultrasonic emulsification of tetradecane and crosslinking of the chitosan shell in aqueous solutions of acetic acid. The concentration of acetic acid in aqueous solutions of chitosan was varied from 0.2% to 25% v/v. The effect of acetic acid concentration and size of the individual microcapsules on the strength was probed. The deformations and forces required to rupture the microcapsules were also measured. Three dimensional deformations of microcapsules under large applied loads were obtained by the combination of Laser Scanning Confocal Microscopy (LSCM) with Atomic Force Microscopy (AFM). The stiffness, and hence the modulus, of the microcapsules was found to decrease with an increase in size with the average stiffness ranging from 82 to 111 mN m-1 and average Young's modulus ranging from 0.4 to 6.5 MPa. The forces required to rupture the microcapsules varied from 150 to 250 nN with deformations of the microcapsules up to 62 to 110% relative to their radius, respectively. Three dimensional images obtained using laser scanning confocal microscopy showed that the microcapsules retained their structure and shape after being subjected to large deformations and subsequent removal of the loads. Based on the above observations, the oil filled chitosan crosslinked microcapsules are an ideal choice for use in the food and pharmaceutical industries as they would be able to withstand the process conditions encountered.

Coherent Raman Scattering Microscopy in Biology and Medicine

Science.gov (United States)

Zhang, Chi; Zhang, Delong; Cheng, Ji-Xin

2016-01-01

Advancements in coherent Raman scattering (CRS) microscopy have enabled label-free visualization and analysis of functional, endogenous biomolecules in living systems. When compared with spontaneous Raman microscopy, a key advantage of CRS microscopy is the dramatic improvement in imaging speed, which gives rise to real-time vibrational imaging of live biological samples. Using molecular vibrational signatures, recently developed hyperspectral CRS microscopy has improved the readout of chemical information available from CRS images. In this article, we review recent achievements in CRS microscopy, focusing on the theory of the CRS signal-to-noise ratio, imaging speed, technical developments, and applications of CRS imaging in bioscience and clinical settings. In addition, we present possible future directions that the use of this technology may take. PMID:26514285

Magnetic elements for switching magnetization magnetic force microscopy tips

International Nuclear Information System (INIS)

Cambel, V.; Elias, P.; Gregusova, D.; Martaus, J.; Fedor, J.; Karapetrov, G.; Novosad, V.

2010-01-01

Using combination of micromagnetic calculations and magnetic force microscopy (MFM) imaging we find optimal parameters for novel magnetic tips suitable for switching magnetization MFM. Switching magnetization MFM is based on two-pass scanning atomic force microscopy with reversed tip magnetization between the scans. Within the technique the sum of the scanned data with reversed tip magnetization depicts local atomic forces, while their difference maps the local magnetic forces. Here we propose the design and calculate the magnetic properties of tips suitable for this scanning probe technique. We find that for best performance the spin-polarized tips must exhibit low magnetic moment, low switching fields, and single-domain state at remanence. The switching field of such tips is calculated and optimum shape of the Permalloy elements for the tips is found. We show excellent correspondence between calculated and experimental results for Py elements.

High resolution, high speed ultrahigh vacuum microscopy

International Nuclear Information System (INIS)

Poppa, Helmut

2004-01-01

The history and future of transmission electron microscopy (TEM) is discussed as it refers to the eventual development of instruments and techniques applicable to the real time in situ investigation of surface processes with high resolution. To reach this objective, it was necessary to transform conventional high resolution instruments so that an ultrahigh vacuum (UHV) environment at the sample site was created, that access to the sample by various in situ sample modification procedures was provided, and that in situ sample exchanges with other integrated surface analytical systems became possible. Furthermore, high resolution image acquisition systems had to be developed to take advantage of the high speed imaging capabilities of projection imaging microscopes. These changes to conventional electron microscopy and its uses were slowly realized in a few international laboratories over a period of almost 40 years by a relatively small number of researchers crucially interested in advancing the state of the art of electron microscopy and its applications to diverse areas of interest; often concentrating on the nucleation, growth, and properties of thin films on well defined material surfaces. A part of this review is dedicated to the recognition of the major contributions to surface and thin film science by these pioneers. Finally, some of the important current developments in aberration corrected electron optics and eventual adaptations to in situ UHV microscopy are discussed. As a result of all the path breaking developments that have led to today's highly sophisticated UHV-TEM systems, integrated fundamental studies are now possible that combine many traditional surface science approaches. Combined investigations to date have involved in situ and ex situ surface microscopies such as scanning tunneling microscopy/atomic force microscopy, scanning Auger microscopy, and photoemission electron microscopy, and area-integrating techniques such as x-ray photoelectron

Nanomorphology of polythiophene–fullerene bulk-heterojunction films investigated by structured illumination optical imaging and time-resolved confocal microscopy

International Nuclear Information System (INIS)

Hao, X-T; Hirvonen, L M; Smith, T A

2013-01-01

Structured illumination microscopy (SIM) and time-resolved confocal fluorescence microscopy are applied to investigate the nanomorphology of thin films comprising typical blends of the conjugated polymer, poly (3-hexylthiophene) (P3HT), and [6, 6]-phenyl C 61 -butyric acid methyl ester (PCBM), used for organic photovoltaic applications. SIM provides evidence for the presence of a thin emissive region around the crystalline regions of PCBM and at the tips of rod-like domains. The time-resolved measurements show that the emission surrounding the PCBM rods is longer lived than the bulk of the film. The two modes of microscopy provide complementary evidence indicating that electron–hole separation is inhibited between the polymer and the large PCBM-rich domains in these regions. We show here that structured illumination microscopy is a viable method of gaining additional information from these photovoltaic materials, despite their weak emission. (paper)

Preventing probe induced topography correlated artifacts in Kelvin Probe Force Microscopy

NARCIS (Netherlands)

Polak, L.; Wijngaarden, Rinke J.

2016-01-01

Kelvin Probe Force Microscopy (KPFM) on samples with rough surface topography can be hindered by topography correlated artifacts. We show that, with the proper experimental configuration and using homogeneously metal coated probes, we are able to obtain amplitude modulation (AM) KPFM results on a

Investigation of Nematode Diversity using Scanning Electron Microscopy and Fluorescent Microscopy

Science.gov (United States)

Seacor, Taylor; Howell, Carina

2013-03-01

Nematode worms account for the vast majority of the animals in the biosphere. They are colossally important to global public health as parasites, and to agriculture both as pests and as beneficial inhabitants of healthy soil. Amphid neurons are the anterior chemosensory neurons in nematodes, mediating critical behaviors including chemotaxis and mating. We are examining the cellular morphology and external anatomy of amphid neurons, using fluorescence microscopy and scanning electron microscopy, respectively, of a wide range of soil nematodes isolated in the wild. We use both classical systematics (e.g. diagnostic keys) and molecular markers (e.g. ribosomal RNA) to classify these wild isolates. Our ultimate aim is to build a detailed anatomical database in order to dissect genetic pathways of neuronal development and function across phylogeny and ecology. Research supported by NSF grants 092304, 0806660, 1058829 and Lock Haven University FPDC grants

Localization and biosynthesis of aminopeptidase N in pig fetal small intestine

DEFF Research Database (Denmark)

Danielsen, E M; Niels-Christiansen, L L; Hansen, Gert Helge

1995-01-01

BACKGROUND & AIMS: Little is known about the expression of brush border enzymes in fetal enterocytes. The aim of this study was to describe the localization and biosynthesis of porcine fetal aminopeptidase N. METHODS: This study was performed using histochemistry and immunoelectron microscopy......, and large vacuoles in the apical cytoplasm. The transient high mannose-glycosylated form of fetal aminopeptidase N was processed to the mature complex-glycosylated form at a markedly slower rate than the enzyme in adult intestine. Likewise, dimerization occurred slowly compared with the adult form...... of aminopeptidase N, and it took place mainly after the Golgi-associated complex glycosylation. The enzyme had a biphasic appearance in the Mg(2+)-precipitated and microvillar fractions, indicating that the bulk of newly made aminopeptidase N is transported to the brush border membrane before appearing...

Multispectral photoacoustic microscopy of lipids using a pulsed supercontinuum laser.

Science.gov (United States)

Buma, Takashi; Conley, Nicole C; Choi, Sang Won

2018-01-01

We demonstrate optical resolution photoacoustic microscopy (OR-PAM) of lipid-rich tissue between 1050-1714 nm using a pulsed supercontinuum laser based on a large-mode-area photonic crystal fiber. OR-PAM experiments of lipid-rich samples show the expected optical absorption peaks near 1210 and 1720 nm. These results show that pulsed supercontinuum lasers are promising for OR-PAM applications such as label-free histology of lipid-rich tissue and imaging small animal models of disease.

Hybrid Microscopy: Enabling Inexpensive High-Performance Imaging through Combined Physical and Optical Magnifications.

Science.gov (United States)

Zhang, Yu Shrike; Chang, Jae-Byum; Alvarez, Mario Moisés; Trujillo-de Santiago, Grissel; Aleman, Julio; Batzaya, Byambaa; Krishnadoss, Vaishali; Ramanujam, Aishwarya Aravamudhan; Kazemzadeh-Narbat, Mehdi; Chen, Fei; Tillberg, Paul W; Dokmeci, Mehmet Remzi; Boyden, Edward S; Khademhosseini, Ali

2016-03-15

To date, much effort has been expended on making high-performance microscopes through better instrumentation. Recently, it was discovered that physical magnification of specimens was possible, through a technique called expansion microscopy (ExM), raising the question of whether physical magnification, coupled to inexpensive optics, could together match the performance of high-end optical equipment, at a tiny fraction of the price. Here we show that such "hybrid microscopy" methods--combining physical and optical magnifications--can indeed achieve high performance at low cost. By physically magnifying objects, then imaging them on cheap miniature fluorescence microscopes ("mini-microscopes"), it is possible to image at a resolution comparable to that previously attainable only with benchtop microscopes that present costs orders of magnitude higher. We believe that this unprecedented hybrid technology that combines expansion microscopy, based on physical magnification, and mini-microscopy, relying on conventional optics--a process we refer to as Expansion Mini-Microscopy (ExMM)--is a highly promising alternative method for performing cost-effective, high-resolution imaging of biological samples. With further advancement of the technology, we believe that ExMM will find widespread applications for high-resolution imaging particularly in research and healthcare scenarios in undeveloped countries or remote places.

Cell junction protein armadillo repeat gene deleted in velo-cardio-facial syndrome is expressed in the skin and colocalizes with autoantibodies of patients affected by a new variant of endemic pemphigus foliaceus in Colombia.

Science.gov (United States)

Abreu-Velez, Ana Maria; Yi, Hong; Howard, Michael S

2017-10-01

We previously described a new variant of endemic pemphigus foliaceus in El Bagre, Colombia, South America (El Bagre-EPF, or pemphigus Abreu-Manu). El Bagre-EPF differs from other types of EPF clinically, epidemiologically, immunologically and in its target antigens. We reported the presence of patient autoantibodies colocalizing with armadillo repeat gene deleted in velo-cardio-facial syndrome (ARVCF), a catenin cell junction protein colocalizing with El Bagre-EPF autoantibodies in the heart and within pilosebaceous units along their neurovascular supply routes. Here we investigate the presence of ARVCF in skin and its possibility as a cutaneous El Bagre-EPF antigen. We used a case-control study, testing sera of 45 patients and 45 controls via direct and indirect immunofluorescence (DIF/IIF), confocal microscopy, immunoelectron microscopy and immunoblotting for the presence of ARVCF and its relationship with El Bagre-EPF autoantibodies in the skin. We also immunoadsorbed samples with desmoglein 1 (Dsg1) ectodomain (El Bagre-EPF antigen) by incubating with the positive ARVCF samples from DIF and IIF. ARVCF was expressed in all the samples from the cases and controls. Immunoadsorption with Dsg1 on positive ARVCF immunofluorescence DIF/IIF cases showed that the immune response was present against non-desmoglein 1 antigen(s). Overall, 40/45 patients showed colocalization of their autoantibodies with ARVCF in the epidermis; no controls from the endemic area displayed colocalization. We demonstrate that ARVCF is expressed in many areas of human skin, and colocalizes with the majority of El Bagre-EPF autoantibodies as a putative antigen.

Photoacoustic microscopy of bilirubin in tissue phantoms

Science.gov (United States)

Zhou, Yong; Zhang, Chi; Yao, Da-Kang; Wang, Lihong V.

2012-12-01

Determining both bilirubin's concentration and its spatial distribution are important in disease diagnosis. Here, for the first time, we applied quantitative multiwavelength photoacoustic microscopy (PAM) to detect bilirubin concentration and distribution simultaneously. By measuring tissue-mimicking phantoms with different bilirubin concentrations, we showed that the root-mean-square error of prediction has reached 0.52 and 0.83 mg/dL for pure bilirubin and for blood-mixed bilirubin detection (with 100% oxygen saturation), respectively. We further demonstrated the capability of the PAM system to image bilirubin distribution both with and without blood. Finally, by underlaying bilirubin phantoms with mouse skins, we showed that bilirubin can be imaged with consistent accuracy down to >400 μm in depth. Our results show that PAM has potential for noninvasive bilirubin monitoring in vivo, as well as for further clinical applications.

Applications of microscopy in Salmonella research.

Science.gov (United States)

Malt, Layla M; Perrett, Charlotte A; Humphrey, Suzanne; Jepson, Mark A

2015-01-01

Salmonella enterica is a Gram-negative enteropathogen that can cause localized infections, typically resulting in gastroenteritis, or systemic infection, e.g., typhoid fever, in humans and many other animals. Understanding the mechanisms by which Salmonella induces disease has been the focus of intensive research. This has revealed that Salmonella invasion requires dynamic cross-talk between the microbe and host cells, in which bacterial adherence rapidly leads to a complex sequence of cellular responses initiated by proteins translocated into the host cell by a type 3 secretion system. Once these Salmonella-induced responses have resulted in bacterial invasion, proteins translocated by a second type 3 secretion system initiate further modulation of cellular activities to enable survival and replication of the invading pathogen. Elucidation of the complex and highly dynamic pathogen-host interactions ultimately requires analysis at the level of single cells and single infection events. To achieve this goal, researchers have applied a diverse range of microscopy techniques to analyze Salmonella infection in models ranging from whole animal to isolated cells and simple eukaryotic organisms. For example, electron microscopy and high-resolution light microscopy techniques such as confocal microscopy can reveal the precise location of Salmonella and its relationship to cellular components. Widefield light microscopy is a simpler approach with which to study the interaction of bacteria with host cells and often has advantages for live cell imaging, enabling detailed analysis of the dynamics of infection and cellular responses. Here we review the use of imaging techniques in Salmonella research and compare the capabilities of different classes of microscope to address specific types of research question. We also provide protocols and notes on some microscopy techniques used routinely in our own research.

Fluorescence microscopy for the characterization of structural integrity

Science.gov (United States)

Street, Kenneth W.; Leonhardt, Todd A.

1991-01-01

The absorption characteristics of light and the optical technique of fluorescence microscopy for enhancing metallographic interpretation are presented. Characterization of thermally sprayed coatings by optical microscopy suffers because of the tendency for misidentification of the microstructure produced by metallographic preparation. Gray scale, in bright field microscopy, is frequently the only means of differentiating the actual structural details of porosity, cracking, and debonding of coatings. Fluorescence microscopy is a technique that helps to distinguish the artifacts of metallographic preparation (pullout, cracking, debonding) from the microstructure of the specimen by color contrasting structural differences. Alternative instrumentation and the use of other dye systems are also discussed. The combination of epoxy vacuum infiltration with fluorescence microscopy to verify microstructural defects is an effective means to characterize advanced materials and to assess structural integrity.

Optofluidic time-stretch microscopy: recent advances

Science.gov (United States)

Lei, Cheng; Nitta, Nao; Ozeki, Yasuyuki; Goda, Keisuke

2018-04-01

Flow cytometry is an indispensable method for valuable applications in numerous fields such as immunology, pathology, pharmacology, molecular biology, and marine biology. Optofluidic time-stretch microscopy is superior to conventional flow cytometry methods for its capability to acquire high-quality images of single cells at a high-throughput exceeding 10,000 cells per second. This makes it possible to extract copious information from cellular images for accurate cell detection and analysis with the assistance of machine learning. Optofluidic time-stretch microscopy has proven its effectivity in various applications, including microalga-based biofuel production, evaluation of thrombotic disorders, as well as drug screening and discovery. In this review, we discuss the principles and recent advances of optofluidic time-stretch microscopy.

Electron Microscopy Center (EMC)

Data.gov (United States)

Federal Laboratory Consortium — The Electron Microscopy Center (EMC) at Argonne National Laboratory develops and maintains unique capabilities for electron beam characterization and applies those...

Colloidal graphite/graphene nanostructures using collagen showing enhanced thermal conductivity

Science.gov (United States)

Bhattacharya, Soumya; Dhar, Purbarun; Das, Sarit K; Ganguly, Ranjan; Webster, Thomas J; Nayar, Suprabha

2014-01-01

In the present study, the exfoliation of natural graphite (GR) directly to colloidal GR/graphene (G) nanostructures using collagen (CL) was studied as a safe and scalable process, akin to numerous natural processes and hence can be termed “biomimetic”. Although the exfoliation and functionalization takes place in just 1 day, it takes about 7 days for the nano GR/G flakes to stabilize. The predominantly aromatic residues of the triple helical CL forms its own special micro and nanoarchitecture in acetic acid dispersions. This, with the help of hydrophobic and electrostatic forces, interacts with GR and breaks it down to nanostructures, forming a stable colloidal dispersion. Surface enhanced Raman spectroscopy, X-ray diffraction, photoluminescence, fluorescence, and X-ray photoelectron spectroscopy of the colloid show the interaction between GR and CL on day 1 and 7. Differential interference contrast images in the liquid state clearly reveal how the GR flakes are entrapped in the CL fibrils, with a corresponding fluorescence image showing the intercalation of CL within GR. Atomic force microscopy of graphene-collagen coated on glass substrates shows an average flake size of 350 nm, and the hexagonal diffraction pattern and thickness contours of the G flakes from transmission electron microscopy confirm ≤ five layers of G. Thermal conductivity of the colloid shows an approximate 17% enhancement for a volume fraction of less than approximately 0.00005 of G. Thus, through the use of CL, this new material and process may improve the use of G in terms of biocompatibility for numerous medical applications that currently employ G, such as internally controlled drug-delivery assisted thermal ablation of carcinoma cells. PMID:24648728

Colloidal graphite/graphene nanostructures using collagen showing enhanced thermal conductivity.

Science.gov (United States)

Bhattacharya, Soumya; Dhar, Purbarun; Das, Sarit K; Ganguly, Ranjan; Webster, Thomas J; Nayar, Suprabha

2014-01-01

In the present study, the exfoliation of natural graphite (GR) directly to colloidal GR/graphene (G) nanostructures using collagen (CL) was studied as a safe and scalable process, akin to numerous natural processes and hence can be termed "biomimetic". Although the exfoliation and functionalization takes place in just 1 day, it takes about 7 days for the nano GR/G flakes to stabilize. The predominantly aromatic residues of the triple helical CL forms its own special micro and nanoarchitecture in acetic acid dispersions. This, with the help of hydrophobic and electrostatic forces, interacts with GR and breaks it down to nanostructures, forming a stable colloidal dispersion. Surface enhanced Raman spectroscopy, X-ray diffraction, photoluminescence, fluorescence, and X-ray photoelectron spectroscopy of the colloid show the interaction between GR and CL on day 1 and 7. Differential interference contrast images in the liquid state clearly reveal how the GR flakes are entrapped in the CL fibrils, with a corresponding fluorescence image showing the intercalation of CL within GR. Atomic force microscopy of graphene-collagen coated on glass substrates shows an average flake size of 350 nm, and the hexagonal diffraction pattern and thickness contours of the G flakes from transmission electron microscopy confirm ≤ five layers of G. Thermal conductivity of the colloid shows an approximate 17% enhancement for a volume fraction of less than approximately 0.00005 of G. Thus, through the use of CL, this new material and process may improve the use of G in terms of biocompatibility for numerous medical applications that currently employ G, such as internally controlled drug-delivery assisted thermal ablation of carcinoma cells.

Electron microscopy of intermediate filaments: teaming up with atomic force and confocal laser scanning microscopy.

Science.gov (United States)

Kreplak, Laurent; Richter, Karsten; Aebi, Ueli; Herrmann, Harald

2008-01-01

Intermediate filaments (IFs) were originally discovered and defined by electron microscopy in myoblasts. In the following it was demonstrated and confirmed that they constitute, in addition to microtubules and microfilaments, a third independent, general filament system in the cytoplasm of most metazoan cells. In contrast to the other two systems, IFs are present in cells in two principally distinct cytoskeletal forms: (i) extended and free-running filament arrays in the cytoplasm that are integrated into the cytoskeleton by associated proteins of the plakin type; and (ii) a membrane- and chromatin-bound thin 'lamina' of a more or less regular network of interconnected filaments made from nuclear IF proteins, the lamins, which differ in several important structural aspects from cytoplasmic IF proteins. In man, more than 65 genes code for distinct IF proteins that are expressed during embryogenesis in various routes of differentiation in a tightly controlled manner. IF proteins exhibit rather limited sequence identity implying that the different types of IFs have distinct biochemical properties. Hence, to characterize the structural properties of the various IFs, in vitro assembly regimes have been developed in combination with different visualization methods such as transmission electron microscopy of fixed and negatively stained samples as well as methods that do not use staining such as scanning transmission electron microscopy (STEM) and cryoelectron microscopy as well as atomic force microscopy. Moreover, with the generation of both IF-type specific antibodies and chimeras of fluorescent proteins and IF proteins, it has become possible to investigate the subcellular organization of IFs by correlative fluorescence and electron microscopic methods. The combination of these powerful methods should help to further develop our understanding of nuclear architecture, in particular how nuclear subcompartments are organized and in which way lamins are involved.

Differentiating the two main histologic categories of fibroadenoma tissue from normal breast tissue by using multiphoton microscopy.

Science.gov (United States)

Nie, Y T; Wu, Y; Fu, F M; Lian, Y E; Zhuo, S M; Wang, C; Chen, J X

2015-04-01

Multiphoton microscopy has become a novel biological imaging technique that allows cellular and subcellular microstructure imaging based on two-photon excited fluorescence and second harmonic generation. In this work, we used multiphoton microscopy to obtain the high-contrast images of human normal breast tissue and two main histologic types of fibroadenoma (intracanalicular, pericanalicular). Moreover, quantitative image analysis was performed to characterize the changes of collagen morphology (collagen content, collagen orientation). The results show that multiphoton microscopy combined with quantitative method has the ability to identify the characteristics of fibroadenoma including changes of the duct architecture and collagen morphology in stroma. With the advancement of multiphoton microscopy, we believe that the technique has great potential to be a real-time histopathological diagnostic tool for intraoperative detection of fibroadenoma in the future. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

Microstructure of NiTi orthodontic wires observations using transmission electron microscopy

OpenAIRE

Ferčec, J.; Jenko, D.; Buchmeister, B.; Rojko, F.; Budič, B.; Kosec, B.; Rudolf, R.

2014-01-01

This work presents the results of the microstructure observation of six different types of NiTi orthodontic wires by using Transmission Electron Microscopy (TEM). Within these analyses the chemical compositions of each wire were observed in different places by applying the EDS detector. Namely, the chemical composition in the orthodontic wires is very important because it shows the dependence between the phase temperatures and mechanical properties. Microstructure observations showed that ort...

Light microscopy - Methods and protocols

Directory of Open Access Journals (Sweden)

CarloAlberto Redi

2011-11-01

Full Text Available The first part of the book (six chapters is devoted to some selected applications of bright-field microscopy while the second part (eight chapters to some fluorescence microscopy studies. Both animal and plant biology investigations are presented covering multiple fields like immunology, cell signaling, cancer biology and, surprisingly to me, ecology. This chapter is titled: Light microscopy in aquatic ecology: Methods for plankton communities studies and it is due to Maria Carolina S. Soares and colleagues from the Laboratory of Aquatic Ecology, Dept. of Biology, Federal University of Juiz de Fora (Brazil. Here they present methods to quantify the different component of planktonic communities in a step-by-step manner so that virus, bacteria, algae and animals pertaining to different taxa can be recognized and the contribution they made to the plankton composition evaluated. It descends that even how the plankton composition is changing due to environmental variations can be accurately determined....

The influence of surface topography on Kelvin probe force microscopy

International Nuclear Information System (INIS)

Sadewasser, S; Leendertz, C; Streicher, F; Lux-Steiner, M Ch

2009-01-01

Long-range electrostatic forces govern the imaging mechanism in electrostatic force microscopy as well as in Kelvin probe force microscopy. To improve the analysis of such images, simulations of the electrostatic field distribution have been performed in the past using a flat surface and a cone-shaped tip. However, the electrostatic field distribution between a tip and a sample depends strongly on the surface topography, which has been neglected in previous studies. It is therefore of general importance to study the influence of sample topography features on Kelvin probe force microscopy images, which we address here by performing finite element simulations. We show how the surface potential measurement is influenced by surface steps and surface grooves, considering potential variations in the form of a potential peak and a potential step. The influence of the topography on the measurement of the surface potential is found to be rather small compared to a typical experimental resolution. Surprisingly, in the case of a coinciding topography and potential step an improvement of the potential profile due to the inclusion of the topography is observed. Finally, based on the obtained results, suggestions for the realization of KPFM measurement are given.

Confocal microscopy for astrocyte in vivo imaging: Recycle and reuse in microscopy

Science.gov (United States)

Pérez-Alvarez, Alberto; Araque, Alfonso; Martín, Eduardo D.

2013-01-01

In vivo imaging is one of the ultimate and fundamental approaches for the study of the brain. Two-photon laser scanning microscopy (2PLSM) constitutes the state-of-the-art technique in current neuroscience to address questions regarding brain cell structure, development and function, blood flow regulation and metabolism. This technique evolved from laser scanning confocal microscopy (LSCM), which impacted the field with a major improvement in image resolution of live tissues in the 1980s compared to widefield microscopy. While nowadays some of the unparalleled features of 2PLSM make it the tool of choice for brain studies in vivo, such as the possibility to image deep within a tissue, LSCM can still be useful in this matter. Here we discuss the validity and limitations of LSCM and provide a guide to perform high-resolution in vivo imaging of the brain of live rodents with minimal mechanical disruption employing LSCM. We describe the surgical procedure and experimental setup that allowed us to record intracellular calcium variations in astrocytes evoked by sensory stimulation, and to monitor intact neuronal dendritic spines and astrocytic processes as well as blood vessel dynamics. Therefore, in spite of certain limitations that need to be carefully considered, LSCM constitutes a useful, convenient, and affordable tool for brain studies in vivo. PMID:23658537

Characterization of muscle contraction with second harmonic generation microscopy

Science.gov (United States)

Prent, Nicole

Muscle cells have the ability to change length and generate force due to orchestrated action of myosin nanomotors that cause sliding of actin filaments along myosin filaments in the sarcomeres, the fundamental contractile units, of myocytes. The correlated action of hundreds of sarcomeres is needed to produce the myocyte contractions. This study probes the molecular structure of the myofilaments and investigates the movement correlations between sarcomeres during contraction. In this study, second harmonic generation (SHG) microscopy is employed for imaging striated myocytes. Myosin filaments in striated myocytes inherently have a nonzero second-order susceptibility, [special characters omitted] and therefore generate efficient SHG. Employing polarization-in polarization-out (PIPO) SHG microscopy allows for the accurate determination of the characteristic ratio, [special characters omitted] in birefringent myocytes, which describes the structure of the myosin filament. Analysis shows that the b value at the centre of the myosin filament, where the nonlinear dipoles are better aligned, is slightly lower than the value at the edges of the filament, where there is more disorder in orientation of the nonlinear dipoles from the myosin heads. Forced stretching of myocytes resulted in an SHG intensity increase with the elongation of the sarcomere. SHG microscopy captured individual sarcomeres during contraction, allowing for the measurement of sarcomere length (SL) and SHG intensity (SI) fluctuations. The fluctuations also revealed higher SHG intensity in elongated sarcomeres. The sarcomere synchronization model (SSM) for contracting and quiescent myocytes was developed, and experimentally verified for three cases (isolated cardiomyocyte, embryonic chicken cardiomyocyte, and larva myocyte). During contraction, the action of SLs and SIs between neighbouring sarcomeres partially correlated, whereas in quiescent myocytes the SLs show an anti-correlation and the SIs have no

System analysis of force feedback microscopy

International Nuclear Information System (INIS)

Rodrigues, Mario S.; Costa, Luca; Chevrier, Joël; Comin, Fabio

2014-01-01

It was shown recently that the Force Feedback Microscope (FFM) can avoid the jump-to-contact in Atomic force Microscopy even when the cantilevers used are very soft, thus increasing force resolution. In this letter, we explore theoretical aspects of the associated real time control of the tip position. We take into account lever parameters such as the lever characteristics in its environment, spring constant, mass, dissipation coefficient, and the operating conditions such as controller gains and interaction force. We show how the controller parameters are determined so that the FFM functions at its best and estimate the bandwidth of the system under these conditions

System analysis of force feedback microscopy

Energy Technology Data Exchange (ETDEWEB)

Rodrigues, Mario S. [CFMC/Dep. de Física, Universidade de Lisboa, Campo Grande, 1749-016 Lisboa (Portugal); Costa, Luca [European Synchrotron Radiation Facility, 6 rue Jules Horowitz BP 220, 38043 Grenoble Cedex (France); Université Joseph Fourier BP 53, 38041 Grenoble Cedex 9 (France); Chevrier, Joël [European Synchrotron Radiation Facility, 6 rue Jules Horowitz BP 220, 38043 Grenoble Cedex (France); Université Grenoble Alpes, Inst NEEL, F-38042 Grenoble (France); CNRS, Inst NEEL, F-38042 Grenoble (France); Comin, Fabio [European Synchrotron Radiation Facility, 6 rue Jules Horowitz BP 220, 38043 Grenoble Cedex (France)

2014-02-07

It was shown recently that the Force Feedback Microscope (FFM) can avoid the jump-to-contact in Atomic force Microscopy even when the cantilevers used are very soft, thus increasing force resolution. In this letter, we explore theoretical aspects of the associated real time control of the tip position. We take into account lever parameters such as the lever characteristics in its environment, spring constant, mass, dissipation coefficient, and the operating conditions such as controller gains and interaction force. We show how the controller parameters are determined so that the FFM functions at its best and estimate the bandwidth of the system under these conditions.

French Society of Microscopies, 11. Colloquium. SFM Paris 2009. Compilation of summaries

International Nuclear Information System (INIS)

2009-06-01

The 11. conference of the SFM (French Society of Microscopies), held in Paris in 2009, was divided into 14 symposiums, 4 GN-MEBA symposiums, and 10 workshops. The titles of the symposiums are: homage to Nicolas Boisset, advanced microscopies, alternative microscopies, new optical and plasmonic imaging microscopies, dynamic and quantitative microscopy of the living matter, photonic and correlative electronic microscopy, near field microscopy, molecular and cellular electronic cryo-microscopy, cellular compartmentation and dynamics (CFPU), microscopy and materials, dynamical microscopy in materials science, minerals/bio-minerals and environment, structure and properties of nano-materials, sub-eV and sub-nm chemical bonds imaging. The titles of the GN-MEBA symposiums are: microscopy and metals, microscopy and minerals, microscopy and living beings, microscopy and new materials. The titles of the workshops are: Correlative Light and Electron Microscopy (CLEM), Cryo and electronic tomography in cellular biology, Cryo electronic microscopy of vitreous sections (CEMOVIS), Atomic Force Microscopy (AFM), ULTRASTEM, Digital Micrograph programming, Cryo-Microscopy and molecular tomography, Cryo-ultra-microtomy and immuno-marking, FIB, ASTAR(EBSD-MET) - rapid mapping of crystalline orientations and phases

Scanning probe recognition microscopy investigation of tissue scaffold properties

Science.gov (United States)

Fan, Yuan; Chen, Qian; Ayres, Virginia M; Baczewski, Andrew D; Udpa, Lalita; Kumar, Shiva

2007-01-01

Scanning probe recognition microscopy is a new scanning probe microscopy technique which enables selective scanning along individual nanofibers within a tissue scaffold. Statistically significant data for multiple properties can be collected by repetitively fine-scanning an identical region of interest. The results of a scanning probe recognition microscopy investigation of the surface roughness and elasticity of a series of tissue scaffolds are presented. Deconvolution and statistical methods were developed and used for data accuracy along curved nanofiber surfaces. Nanofiber features were also independently analyzed using transmission electron microscopy, with results that supported the scanning probe recognition microscopy-based analysis. PMID:18203431

Novel insights into pericarp, protein body globoids of aleurone layer, starchy granules of three cereals gained using atomic force microscopy and environmental scanning electronic microscopy.

Science.gov (United States)

Antonini, Elena; Zara, Carolina; Valentini, Laura; Gobbi, Pietro; Ninfali, Paolino; Menotta, Michele

2018-02-05

In this study, we applied Environmental Scanning Electron Microscopy-Energy Dispersive Spectroscopy (ESEM-EDS) and Atomic Force Microscopy (AFM) analysis to three different cereal caryopses: barley, oat and einkorn wheat. The morphological structures, chemical elemental composition and surface characteristics of the three cereals were described. Regarding the morphology, barley showed the thickest pericarp, providing a strong barrier digestion and absorption of nutrients. The aleurone layer of each cereal type contained protein body globoids within its cells. Large type-A and small type-B starchy granules were revealed in the endosperm of barley and einkorn wheat, whereas irregular starchy granules were found in oats. The starchy granule elemental composition, detected by ESEM-EDS, was rather homogenous in the three cereals, whereas the pericarp and protein body globoids showed heterogeneity. In the protein body globoids, oats showed higher P and K concentrations than barley and einkorn wheat. Regarding the topographic profiles, detected by AFM, einkorn wheat starchy granules showed a surface profile that differed significantly from that of oats and barley, which were quite similar to one another. The present work provides insights into the morphological and chemical makeup of the three grains shedding light on the higher bio-accessibility of einkorn wheat nutrients compared to barley and oats, providing important suggestions for human nutrition and technological standpoints.

Novel insights into pericarp, protein body globoids of aleurone layer, starchy granules of three cereals gained using atomic force microscopy and environmental scanning electronic microscopy

Science.gov (United States)

Antonini, Elena; Zara, Carolina; Valentini, Laura; Gobbi, Pietro; Menotta, Michele

2018-01-01

In this study, we applied Environmental Scanning Electron Microscopy-Energy Dispersive Spectroscopy (ESEM-EDS) and Atomic Force Microscopy (AFM) analysis to three different cereal caryopses: barley, oat and einkorn wheat. The morphological structures, chemical elemental composition and surface characteristics of the three cereals were described. Regarding the morphology, barley showed the thickest pericarp, providing a strong barrier to digestion and absorption of nutrients. The aleurone layer of each cereal type contained protein body globoids within its cells. Large type-A and small type-B starchy granules were revealed in the endosperm of barley and einkorn wheat, whereas irregular starchy granules were found in oats. The starchy granule elemental composition, detected by ESEM-EDS, was rather homogenous in the three cereals, whereas the pericarp and protein body globoids showed heterogeneity. In the protein body globoids, oats showed higher P and K concentrations than barley and einkorn wheat. Regarding the topographic profiles, detected by AFM, einkorn wheat starchy granules showed a surface profile that differed significantly from that of oats and barley, which were quite similar to one another. The present work provides insights into the morphological and chemical makeup of the three grains shedding light on the higher bio-accessibility of einkorn wheat nutrients compared to barley and oats, providing important suggestions for human nutrition and technological standpoints. PMID:29569870

Endomorphin-2 Inhibits the Activity of the Spinoparabrachial Projection Neuron through Presynaptic Mechanisms in the Spinal Dorsal Horn in Rats

Directory of Open Access Journals (Sweden)

Jun-Bin Yin

2018-03-01

Full Text Available Background/Aims: Spinal dorsal horn (SDH is one of the most important regions for analgesia produced by endomorphin-2 (EM2, which has a higher affinity and specificity for the µ-opioid receptor (MOR than morphine. Many studies have focused on substantia gelatinosa (SG, lamina II neurons to elucidate the cellular basis for its antinociceptive effects. However, the complicated types and local circuits of interneurons in the SG make it difficult to understand the real effects of EM2. Therefore, in the present study, we examined the effects of EM2 on projection neurons (PNs in lamina I. Methods: Tracing, immunofluoresence, and immunoelectron methods were used to examine the morphological connections between EM2-immunoreactive (-ir terminals and PNs. By using in vitro whole cell patch clamp recording technique, we investigated the functional effects of EM2 on PNs. Results: EM2-ir afferent terminals directly contacted PNs projecting to the parabrachial nucleus in lamina I. Their synaptic connections were further confirmed by immunoelectron microscopy, most of which were asymmetric synapses. It was found that EM2 had a strong inhibitory effect on the frequency, but not amplitude, of the spontaneous excitatory postsynaptic current (sEPSC of the spinoparabrachial PNs in lamina I, which could be reversed by MOR antagonist CTOP. However, their spontaneous inhibitory postsynaptic current (sIPSC and intrinsic properties were not changed after EM2 application. Conclusion: Applying EM2 to the SDH could produce analgesia through inhibiting the activities of the spinoparabrachial PNs in lamina I by reducing presynaptic neurotransmitters release from the primary afferent terminals.

Nanoscale Laser Terahertz Emission Microscopy

DEFF Research Database (Denmark)

Klarskov, Pernille; Kim, Hyewon; Colvin, Vicki L.

2017-01-01

Laser terahertz emission microscopy (LTEM) has become a powerful tool for studying ultrafast dynamics and local fields in many different types of materials. This technique, which relies on acceleration of charge carriers in a material upon femtosecond excitation, can provide insight into the phys......Laser terahertz emission microscopy (LTEM) has become a powerful tool for studying ultrafast dynamics and local fields in many different types of materials. This technique, which relies on acceleration of charge carriers in a material upon femtosecond excitation, can provide insight...

Confocal filtering in cathodoluminescence microscopy of nanostructures

Science.gov (United States)

Narváez, Angela C.; Weppelman, I. Gerward C.; Moerland, Robert J.; Hoogenboom, Jacob P.; Kruit, Pieter

2014-06-01

Cathodoluminescence (CL) microscopy allows optical characterization of nanostructures at high spatial resolution. At the nanoscale, a main challenge of the technique is related to the background CL generated within the sample substrate. Here, we implement confocal detection of the CL signal to minimize the background contribution to the measurement. Nano-phosphors were used as point sources to evaluate the filtering capabilities of our confocal CL system, obtaining an axial intensity profile with 2.7 μm full width at half maximum for the central peak, in good correspondence with theoretical expectations. Considering the electron interaction volume, we found that the confocal filter becomes effective for electron energies above 20 keV, when using a 25 μm pinhole (0.86 Airy units). To illustrate our approach, we present confocal CL imaging of gold nanowires and triangular shaped plates deposited on an indium-tin oxide covered glass substrate, comparing the images with those obtained in standard unfiltered CL detection. The results show that confocal CL microscopy is a valuable tool for the investigation of nanostructures on highly cathodoluminescent substrates, widely used in biological and optical applications.

Confocal filtering in cathodoluminescence microscopy of nanostructures

Energy Technology Data Exchange (ETDEWEB)

Narváez, Angela C., E-mail: a.c.narvaez@tudelft.nl, E-mail: j.p.hoogenboom@tudelft.nl; Weppelman, I. Gerward C.; Moerland, Robert J.; Hoogenboom, Jacob P., E-mail: a.c.narvaez@tudelft.nl, E-mail: j.p.hoogenboom@tudelft.nl; Kruit, Pieter [Imaging Physics, Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, 2628CJ Delft (Netherlands)

2014-06-23

Cathodoluminescence (CL) microscopy allows optical characterization of nanostructures at high spatial resolution. At the nanoscale, a main challenge of the technique is related to the background CL generated within the sample substrate. Here, we implement confocal detection of the CL signal to minimize the background contribution to the measurement. Nano-phosphors were used as point sources to evaluate the filtering capabilities of our confocal CL system, obtaining an axial intensity profile with 2.7 μm full width at half maximum for the central peak, in good correspondence with theoretical expectations. Considering the electron interaction volume, we found that the confocal filter becomes effective for electron energies above 20 keV, when using a 25 μm pinhole (0.86 Airy units). To illustrate our approach, we present confocal CL imaging of gold nanowires and triangular shaped plates deposited on an indium-tin oxide covered glass substrate, comparing the images with those obtained in standard unfiltered CL detection. The results show that confocal CL microscopy is a valuable tool for the investigation of nanostructures on highly cathodoluminescent substrates, widely used in biological and optical applications.

Elemental mapping in scanning transmission electron microscopy

International Nuclear Information System (INIS)

Allen, L J; D'Alfonso, A J; Lugg, N R; Findlay, S D; LeBeau, J M; Stemmer, S

2010-01-01

We discuss atomic resolution chemical mapping in scanning transmission electron microscopy (STEM) based on core-loss electron energy loss spectroscopy (EELS) and also on energy dispersive X-ray (EDX) imaging. Chemical mapping using EELS can yield counterintuitive results which, however, can be understood using first principles calculations. Experimental chemical maps based on EDX bear out the thesis that such maps are always likely to be directly interpretable. This can be explained in terms of the local nature of the effective optical potential for ionization under those imaging conditions. This is followed by an excursion into the complementary technique of elemental mapping using energy-filtered transmission electron microscopy (EFTEM) in a conventional transmission electron microscope. We will then consider the widely used technique of Z-contrast or high-angle annular dark field (HAADF) imaging, which is based on phonon excitation, where it has recently been shown that intensity variations can be placed on an absolute scale by normalizing the measured intensities to the incident beam. Results, showing excellent agreement between theory and experiment to within a few percent, are shown for Z-contrast imaging from a sample of PbWO 4 .

Photoacoustic microscopy imaging for microneedle drug delivery

Science.gov (United States)

Moothanchery, Mohesh; Seeni, Razina Z.; Xu, Chenjie; Pramanik, Manojit

2018-02-01

The recent development of novel transdermal drug delivery systems (TDDS) using microneedle technology allows micron-sized conduits to be formed within the outermost skin layers attracting keen interest in skin as an interface for localized and systemic delivery of therapeutics. In light of this, researchers are using microneedles as tools to deliver nanoparticle formulations to targeted sites for effective therapy. However, in such studies the use of traditional histological methods are employed for characterization and do not allow for the in vivo visualization of drug delivery mechanism. Hence, this study presents a novel imaging technology to characterize microneedle based nanoparticle delivery systems using optical resolution-photoacoustic microscopy (OR-PAM). In this study in vivo transdermal delivery of gold nanoparticles using microneedles in mice ear and the spatial distribution of the nanoparticles in the tissue was successfully illustrated. Characterization of parameters that are relevant in drug delivery studies such as penetration depth, efficiency of delivered gold nanoparticles were monitored using the system. Photoacoustic microscopy proves an ideal tool for the characterization studies of microneedle properties and the studies shows microneedles as an ideal tool for precise and controlled drug delivery.

Applications of cost-effective spectral imaging microscopy in cancer research

International Nuclear Information System (INIS)

Barber, P R; Vojnovic, B; Atkin, G; Daley, F M; Everett, S A; Wilson, G D; Gilbey, J D

2003-01-01

The application of a cost-effective spectral imager to spatially segmenting absorptive and fluorescent chemical probes on the basis of their spectral characteristics has been demonstrated. The imager comprises a computer-controlled spectrally selective element that allows random access to a bandwidth of 15 nm between 400 and 700 nm. Further, the use of linear un-mixing of the spectral response of a sample at a single pixel has been facilitated using non-negative least squares fitting. Examples are given showing the separation of dye distributions, such as immunohistochemical markers for tumour hypoxia, from multiply stained thin tissue sections, imaged by trans-illumination microscopy. A quantitative study is also presented that shows a correlation between staining intensity and normal versus tumour tissue, and the advantage of reducing the amount of data captured for a particular study is also demonstrated. An example of the application to fluorescence microscopy is also given, showing the separation of green fluorescent protein, Cy3 and Cy5 at a single pixel. The system has been validated against samples of known optical density and of known overlapping combinations of coloured filters. These results confirm the ability of this technique to separate spectral responses that cannot be resolved with conventional colour imaging

Polarizability of DNA Block Copolymer Nanoparticles Observed by Electrostatic Force Microscopy

NARCIS (Netherlands)

Sowwan, Mukhles; Faroun, Maryam; Mentovich, Elad; Ibrahim, Imad; Haboush, Shayma; Alemdaroglu, Fikri Emrah; Kwak, Minseok; Richter, Shachar; Herrmann, Andreas

2010-01-01

In this study, DNA block copolymer (DBC) micelles with a polystyrene (PS) core and a single-stranded (ss) DNA shell were doped with ferrocene (Fc) molecules. Tapping mode atomic force microscopy (AFM) was used to study the morphology of the doped and undoped block copolymer aggregates. We show that

Use of atomic force microscopy and transmission electron microscopy for correlative studies of bacterial capsules.

Science.gov (United States)

Stukalov, Oleg; Korenevsky, Anton; Beveridge, Terry J; Dutcher, John R

2008-09-01

Bacteria can possess an outermost assembly of polysaccharide molecules, a capsule, which is attached to their cell wall. We have used two complementary, high-resolution microscopy techniques, atomic force microscopy (AFM) and transmission electron microscopy (TEM), to study bacterial capsules of four different gram-negative bacterial strains: Escherichia coli K30, Pseudomonas aeruginosa FRD1, Shewanella oneidensis MR-4, and Geobacter sulfurreducens PCA. TEM analysis of bacterial cells using different preparative techniques (whole-cell mounts, conventional embeddings, and freeze-substitution) revealed capsules for some but not all of the strains. In contrast, the use of AFM allowed the unambiguous identification of the presence of capsules on all strains used in the present study, including those that were shown by TEM to be not encapsulated. In addition, the use of AFM phase imaging allowed the visualization of the bacterial cell within the capsule, with a depth sensitivity that decreased with increasing tapping frequency.

Neural plasticity explored by correlative two-photon and electron/SPIM microscopy

Science.gov (United States)

Allegra Mascaro, A. L.; Silvestri, L.; Costantini, I.; Sacconi, L.; Maco, B.; Knott, G. W.; Pavone, F. S.

2013-06-01

Plasticity of the central nervous system is a complex process which involves the remodeling of neuronal processes and synaptic contacts. However, a single imaging technique can reveal only a small part of this complex machinery. To obtain a more complete view, complementary approaches should be combined. Two-photon fluorescence microscopy, combined with multi-photon laser nanosurgery, allow following the real-time dynamics of single neuronal processes in the cerebral cortex of living mice. The structural rearrangement elicited by this highly confined paradigm of injury can be imaged in vivo first, and then the same neuron could be retrieved ex-vivo and characterized in terms of ultrastructural features of the damaged neuronal branch by means of electron microscopy. Afterwards, we describe a method to integrate data from in vivo two-photon fluorescence imaging and ex vivo light sheet microscopy, based on the use of major blood vessels as reference chart. We show how the apical dendritic arbor of a single cortical pyramidal neuron imaged in living mice can be found in the large-scale brain reconstruction obtained with light sheet microscopy. Starting from its apical portion, the whole pyramidal neuron can then be segmented and located in the correct cortical layer. With the correlative approach presented here, researchers will be able to place in a three-dimensional anatomic context the neurons whose dynamics have been observed with high detail in vivo.

Confocal Raman Microscopy; applications in tissue engineering

NARCIS (Netherlands)

van Apeldoorn, Aart A.

2005-01-01

This dissertation describes the use of confocal Raman microscopy and spectroscopy in the field of tissue engineering. Moreover, it describes the combination of two already existing technologies, namely scanning electron microscopy and confocal Raman spectroscopy in one apparatus for the enhancement

Internet-Enabled High-Resolution Brain Mapping and Virtual Microscopy

OpenAIRE

Mikula, Shawn; Trotts, Issac; Stone, James M.; Jones, Edward G.

2007-01-01

Virtual microscopy involves the conversion of histological sections mounted on glass microscope slides to high resolution digital images. Virtual microscopy offers several advantages over traditional microscopy, including remote viewing and data-sharing, annotation, and various forms of data-mining.

Scanning electron microscopy and transmission electron microscopy study of hot-deformed gamma-TiAl-based alloy microstructure.

Science.gov (United States)

Chrapoński, J; Rodak, K

2006-09-01

The aim of this work was to assess the changes in the microstructure of hot-deformed specimens made of alloys containing 46-50 at.% Al, 2 at.% Cr and 2 at.% Nb (and alloying additions such as carbon and boron) with the aid of scanning electron microscopy and transmission electron microscopy techniques. After homogenization and heat treatment performed in order to make diverse lamellae thickness, the specimens were compressed at 1000 degrees C. Transmission electron microscopy examinations of specimens after the compression test revealed the presence of heavily deformed areas with a high density of dislocation. Deformation twins were also observed. Dynamically recrystallized grains were revealed. For alloys no. 2 and no. 3, the recovery and recrystallization processes were more extensive than for alloy no. 1.

[Virtual microscopy in pathology teaching and postgraduate training (continuing education)].

Science.gov (United States)

Sinn, H P; Andrulis, M; Mogler, C; Schirmacher, P

2008-11-01

As with conventional microscopy, virtual microscopy permits histological tissue sections to be viewed on a computer screen with a free choice of viewing areas and a wide range of magnifications. This, combined with the possibility of linking virtual microscopy to E-Learning courses, make virtual microscopy an ideal tool for teaching and postgraduate training in pathology. Uses of virtual microscopy in pathology teaching include blended learning with the presentation of digital teaching slides in the internet parallel to presentation in the histology lab, extending student access to histology slides beyond the lab. Other uses are student self-learning in the Internet, as well as the presentation of virtual slides in the classroom with or without replacing real microscopes. Successful integration of virtual microscopy depends on its embedding in the virtual classroom and the creation of interactive E-learning content. Applications derived from this include the use of virtual microscopy in video clips, podcasts, SCORM modules and the presentation of virtual microscopy using interactive whiteboards in the classroom.

Electron microscopy in metallurgy

International Nuclear Information System (INIS)

Loretto, M.H.

1980-01-01

The aim of this paper is to review briefly the contribution which (TEM) transmission electron microscopy (including high voltage electron microscopy (HVEM)) has made to metallurgy. Since it is straightforward with modern electron microscopes to extract the crystallographic information which provides the basis for any interpretation, the major problem in most metallurgical work lies in assessing how the structure (which TEM has characterised) has arisen and which properties of the specimen can be understood in terms of this structure. Radiation damage, quenching, phase transformations, grain boundaries and plastic deformation have been the main fields in which TEM has contributed significantly. After briefly summarising the role of TEM in each field, examples of recent work will be used to indicate current TEM activity in physical metallurgy. (author)

Scanning tunneling microscopy on iron-chalcogenide superconductor Fe(Se, Te) single crystal

International Nuclear Information System (INIS)

Ukita, R.; Sugimoto, A.; Ekino, T.

2011-01-01

We show scanning tunneling microscopy/spectroscopy (STM/STS) results of Fe(Se, Te). STM topography shows square arrangements of spots with the lattice spacing 0.37 nm. Te and Se atoms are randomly distributed in the STM topography. The STM topography of FeTe exhibits clusters of separated iron atoms. We have investigated the iron-chalcogenide superconductor Fe(Se, Te) using a low-temperature scanning tunneling microscopy/spectroscopy (STM/STS) technique. STM topography at 4.9 K shows clear regular square arrangements of spots with the lattice spacing ∼0.37 nm, from which what we observe are attributed to Se or Te atomic plane. In the topography, brighter and darker atomic spots are randomly distributed, which are most probably due to Te and Se atoms, respectively. For the FeTe compound, the topography exhibits clusters of the bright spots probably arising from separated iron atoms distributing over several Te lattice sites. The STS measurements clarify the existence of the large-size gap with 2Δ = 0.4-0.6 eV.

Integrated Photoacoustic and Fluorescence Confocal Microscopy

OpenAIRE

Wang, Yu; Maslov, Konstantin; Kim, Chulhong; Hu, Song; Wang, Lihong V.

2010-01-01

We have developed a dual-modality imaging system by integrating optical-resolution photoacoustic microscopy and fluorescence confocal microscopy to provide optical absorption and fluorescence contrasts simultaneously. By sharing the same laser source and objective lens, intrinsically registered photoacoustic and fluorescence images are acquired in a single scan. The micrometer resolution allows imaging of both blood and lymphatic vessels down to the capillary level. Simultaneous photoacoustic...

Scanning Ion Conductance Microscopy for Studying Biological Samples

Directory of Open Access Journals (Sweden)

Irmgard D. Dietzel

2012-11-01

Full Text Available Scanning ion conductance microscopy (SICM is a scanning probe technique that utilizes the increase in access resistance that occurs if an electrolyte filled glass micro-pipette is approached towards a poorly conducting surface. Since an increase in resistance can be monitored before the physical contact between scanning probe tip and sample, this technique is particularly useful to investigate the topography of delicate samples such as living cells. SICM has shown its potential in various applications such as high resolution and long-time imaging of living cells or the determination of local changes in cellular volume. Furthermore, SICM has been combined with various techniques such as fluorescence microscopy or patch clamping to reveal localized information about proteins or protein functions. This review details the various advantages and pitfalls of SICM and provides an overview of the recent developments and applications of SICM in biological imaging. Furthermore, we show that in principle, a combination of SICM and ion selective micro-electrodes enables one to monitor the local ion activity surrounding a living cell.

Visualizing radiofrequency-skin interaction using multiphoton microscopy in vivo.

Science.gov (United States)

Tsai, Tsung-Hua; Lin, Sung-Jan; Lee, Woan-Ruoh; Wang, Chun-Chin; Hsu, Chih-Ting; Chu, Thomas; Dong, Chen-Yuan

2012-02-01

Redundant skin laxity is a major feature of aging. Recently, radiofrequency has been introduced for nonablative tissue tightening by volumetric heating of the deep dermis. Despite the wide range of application based on this therapy, the effect of this technique on tissue and the subsequent tissue remodeling have not been investigated in detail. Our objective is to evaluate the potential of non-linear optics, including multiphoton autofluorescence and second harmonic generation (SHG) microscopy, as a non-invasive imaging modality for the real-time study of radiofrequency-tissue interaction. Electro-optical synergy device (ELOS) was used as the radiofrequency source in this study. The back skin of nude mouse was irradiated with radiofrequency at different passes. We evaluated the effect on skin immediately and 1 month after treatment with multiphoton microscopy. Corresponding histology was performed for comparison. We found that SHG is negatively correlated to radiofrequency passes, which means that collagen structural disruption happens immediately after thermal damage. After 1 month of collagen remodeling, SHG signals increased above baseline, indicating that collagen regeneration has occurred. Our findings may explain mechanism of nonablative skin tightening and were supported by histological examinations. Our work showed that monitoring the dermal heating status of RF and following up the detailed process of tissue reaction can be imaged and quantified with multiphoton microscopy non-invasively in vivo. Copyright © 2011. Published by Elsevier Ireland Ltd.

Two-Photon Fluorescence Microscopy Developed for Microgravity Fluid Physics

Science.gov (United States)

Fischer, David G.; Zimmerli, Gregory A.; Asipauskas, Marius

2004-01-01

Recent research efforts within the Microgravity Fluid Physics Branch of the NASA Glenn Research Center have necessitated the development of a microscope capable of high-resolution, three-dimensional imaging of intracellular structure and tissue morphology. Standard optical microscopy works well for thin samples, but it does not allow the imaging of thick samples because of severe degradation caused by out-of-focus object structure. Confocal microscopy, which is a laser-based scanning microscopy, provides improved three-dimensional imaging and true optical sectioning by excluding the out-of-focus light. However, in confocal microscopy, out-of-focus object structure is still illuminated by the incoming beam, which can lead to substantial photo-bleaching. In addition, confocal microscopy is plagued by limited penetration depth, signal loss due to the presence of a confocal pinhole, and the possibility of live-cell damage. Two-photon microscopy is a novel form of laser-based scanning microscopy that allows three-dimensional imaging without many of the problems inherent in confocal microscopy. Unlike one-photon microscopy, it utilizes the nonlinear absorption of two near-infrared photons. However, the efficiency of two-photon absorption is much lower than that of one-photon absorption because of the nonlinear (i.e., quadratic) electric field dependence, so an ultrafast pulsed laser source must typically be employed. On the other hand, this stringent energy density requirement effectively localizes fluorophore excitation to the focal volume. Consequently, two-photon microscopy provides optical sectioning and confocal performance without the need for a signal-limiting pinhole. In addition, there is a reduction in photo-damage because of the longer excitation wavelength, a reduction in background fluorescence, and a 4 increase in penetration depth over confocal methods because of the reduction in Rayleigh scattering.

New developments in transmission electron microscopy for nanotechnology

International Nuclear Information System (INIS)

Wang, Z.L.

2003-01-01

High-resolution transmission electron microscopy (HRTEM) is one of the most powerful tools used for characterizing nanomaterials, and it is indispensable for nanotechnology. This paper reviews some of the most recent developments in electron microscopy techniques for characterizing nanomaterials. The review covers the following areas: in-situ microscopy for studying dynamic shape transformation of nanocrystals; in-situ nanoscale property measurements on the mechanical, electrical and field emission properties of nanotubes/nanowires; environmental microscopy for direct observation of surface reactions; aberration-free angstrom-resolution imaging of light elements (such as oxygen and lithium); high-angle annular-dark-field scanning transmission electron microscopy (STEM); imaging of atom clusters with atomic resolution chemical information; electron holography of magnetic materials; and high-spatial resolution electron energy-loss spectroscopy (EELS) for nanoscale electronic and chemical analysis. It is demonstrated that the picometer-scale science provided by HRTEM is the foundation of nanometer-scale technology. (Abstract Copyright [2003], Wiley Periodicals, Inc.)

In situ transmission electron microscopy and scanning transmission electron microscopy studies of sintering of Ag and Pt nanoparticles

International Nuclear Information System (INIS)

Asoro, M.A.; Ferreira, P.J.; Kovar, D.

2014-01-01

Transmission electron microscopy and scanning transmission electron microscopy studies were conducted in situ on 2–5 nm Pt and 10–40 nm Ag nanoparticles to study mechanisms for sintering and to measure relevant sintering kinetics in nanoscale particles. Sintering between two separated particles was observed to initiate by either (1) diffusion of the particles on the sample support or (2) diffusion of atoms or small clusters of atoms to the neck region between the two particles. After particle contact, the rate of sintering was controlled by atomic surface diffusivity. The surface diffusivity was determined as a function of particle size and temperature from experimental measurements of the rate of neck growth of the particles. The surface diffusivities did not show a strong size effect for the range of particle sizes that were studied. The surface diffusivity for Pt nanoparticles exhibited the expected Arrhenius temperature dependence and did not appear to be sensitive to the presence of surface contaminants. In contrast, the surface diffusivity for Ag nanoparticles was affected by the presence of impurities such as carbon. The diffusivities for Ag nanoparticles were consistent with previous measurements of bulk surface diffusivities for Ag in the presence of C, but were significantly slower than those obtained from pristine Ag

Digital differential confocal microscopy based on spatial shift transformation.

Science.gov (United States)

Liu, J; Wang, Y; Liu, C; Wilson, T; Wang, H; Tan, J

2014-11-01

Differential confocal microscopy is a particularly powerful surface profilometry technique in industrial metrology due to its high axial sensitivity and insensitivity to noise. However, the practical implementation of the technique requires the accurate positioning of point detectors in three-dimensions. We describe a simple alternative based on spatial transformation of a through-focus series of images obtained from a homemade beam scanning confocal microscope. This digital differential confocal microscopy approach is described and compared with the traditional Differential confocal microscopy approach. The ease of use of the digital differential confocal microscopy system is illustrated by performing measurements on a 3D standard specimen. © 2014 The Authors Journal of Microscopy © 2014 Royal Microscopical Society.

Electron microscopy of primary cell cultures in solution and correlative optical microscopy using ASEM

International Nuclear Information System (INIS)

Hirano, Kazumi; Kinoshita, Takaaki; Uemura, Takeshi; Motohashi, Hozumi; Watanabe, Yohei; Ebihara, Tatsuhiko; Nishiyama, Hidetoshi; Sato, Mari; Suga, Mitsuo; Maruyama, Yuusuke; Tsuji, Noriko M.; Yamamoto, Masayuki; Nishihara, Shoko; Sato, Chikara

2014-01-01

Correlative light-electron microscopy of cells in a natural environment of aqueous liquid facilitates high-throughput observation of protein complex formation. ASEM allows the inverted SEM to observe the wet sample from below, while an optical microscope observes it from above quasi-simultaneously. The disposable ASEM dish with a silicon nitride (SiN) film window can be coated variously to realize the primary-culture of substrate-sensitive cells in a few milliliters of culture medium in a stable incubator environment. Neuron differentiation, neural networking, proplatelet-formation and phagocytosis were captured by optical or fluorescence microscopy, and imaged at high resolution by gold-labeled immuno-ASEM with/without metal staining. Fas expression on the cell surface was visualized, correlated to the spatial distribution of F-actin. Axonal partitioning was studied using primary-culture neurons, and presynaptic induction by GluRδ2-N-terminus-linked fluorescent magnetic beads was correlated to the presynaptic-marker Bassoon. Further, megakaryocytes secreting proplatelets were captured, and P-selectins with adherence activity were localized to some of the granules present by immuno-ASEM. The phagocytosis of lactic acid bacteria by dendritic cells was also imaged. Based on these studies, ASEM correlative microscopy promises to allow the study of various mesoscopic-scale dynamics in the near future. - Highlights: • In situ correlative light electron microscopy of samples in open solution by ASEM. • Primary cultures for in-solution CLEM by developing SiN-film coating methods • First visualization of fluorescent magnetic beads in aqueous solution by CLEM. • Presynaptic induction of neurons by GluRδ2-N-terminus-coated beads studied by CLEM. • Axonal partitioning, bacterial phagocytosis, platelet formation imaged by CLEM

Electron microscopy of primary cell cultures in solution and correlative optical microscopy using ASEM

Energy Technology Data Exchange (ETDEWEB)

Hirano, Kazumi; Kinoshita, Takaaki [Laboratory of Cell Biology, Department of Bioinformatics, Faculty of Engineering, Soka University, 1-236 Tangi-machi, Hachioji, Tokyo 192-8577 (Japan); Uemura, Takeshi [Department of Molecular Neurobiology and Pharmacology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Department of Molecular and Cellular Physiology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621 (Japan); Motohashi, Hozumi [Department of Gene Expression Regulation, Institute of Development, Aging and Cancer, Tohoku University, 4-1 Seiryo-cho, Aoba-ku, Sendai 980-8575 (Japan); Watanabe, Yohei; Ebihara, Tatsuhiko [Biomedical Research Institute, National Institute of Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8566 (Japan); Nishiyama, Hidetoshi [JEOL Ltd., 1-2 Musashino 3-chome, Akishima, Tokyo 196-8558 (Japan); Sato, Mari [Biomedical Research Institute, National Institute of Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8566 (Japan); Suga, Mitsuo [JEOL Ltd., 1-2 Musashino 3-chome, Akishima, Tokyo 196-8558 (Japan); Maruyama, Yuusuke; Tsuji, Noriko M. [Biomedical Research Institute, National Institute of Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8566 (Japan); Yamamoto, Masayuki [Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, 2-1 Seiryo-cho, Aoba-ku, Sendai 980-8575 (Japan); Nishihara, Shoko, E-mail: shoko@soka.ac.jp [Laboratory of Cell Biology, Department of Bioinformatics, Faculty of Engineering, Soka University, 1-236 Tangi-machi, Hachioji, Tokyo 192-8577 (Japan); Sato, Chikara, E-mail: ti-sato@aist.go.jp [Biomedical Research Institute, National Institute of Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8566 (Japan)

2014-08-01

Correlative light-electron microscopy of cells in a natural environment of aqueous liquid facilitates high-throughput observation of protein complex formation. ASEM allows the inverted SEM to observe the wet sample from below, while an optical microscope observes it from above quasi-simultaneously. The disposable ASEM dish with a silicon nitride (SiN) film window can be coated variously to realize the primary-culture of substrate-sensitive cells in a few milliliters of culture medium in a stable incubator environment. Neuron differentiation, neural networking, proplatelet-formation and phagocytosis were captured by optical or fluorescence microscopy, and imaged at high resolution by gold-labeled immuno-ASEM with/without metal staining. Fas expression on the cell surface was visualized, correlated to the spatial distribution of F-actin. Axonal partitioning was studied using primary-culture neurons, and presynaptic induction by GluRδ2-N-terminus-linked fluorescent magnetic beads was correlated to the presynaptic-marker Bassoon. Further, megakaryocytes secreting proplatelets were captured, and P-selectins with adherence activity were localized to some of the granules present by immuno-ASEM. The phagocytosis of lactic acid bacteria by dendritic cells was also imaged. Based on these studies, ASEM correlative microscopy promises to allow the study of various mesoscopic-scale dynamics in the near future. - Highlights: • In situ correlative light electron microscopy of samples in open solution by ASEM. • Primary cultures for in-solution CLEM by developing SiN-film coating methods • First visualization of fluorescent magnetic beads in aqueous solution by CLEM. • Presynaptic induction of neurons by GluRδ2-N-terminus-coated beads studied by CLEM. • Axonal partitioning, bacterial phagocytosis, platelet formation imaged by CLEM.

Paleomagnetic Analysis Using SQUID Microscopy

Science.gov (United States)

Weiss, Benjamin P.; Lima, Eduardo A.; Fong, Luis E.; Baudenbacher, Franz J.

2007-01-01

Superconducting quantum interference device (SQUID) microscopes are a new generation of instruments that map magnetic fields with unprecedented spatial resolution and moment sensitivity. Unlike standard rock magnetometers, SQUID microscopes map magnetic fields rather than measuring magnetic moments such that the sample magnetization pattern must be retrieved from source model fits to the measured field data. In this paper, we presented the first direct comparison between paleomagnetic analyses on natural samples using joint measurements from SQUID microscopy and moment magnetometry. We demonstrated that in combination with apriori geologic and petrographic data, SQUID microscopy can accurately characterize the magnetization of lunar glass spherules and Hawaiian basalt. The bulk moment magnitude and direction of these samples inferred from inversions of SQUID microscopy data match direct measurements on the same samples using moment magnetometry. In addition, these inversions provide unique constraints on the magnetization distribution within the sample. These measurements are among the most sensitive and highest resolution quantitative paleomagnetic studies of natural remanent magnetization to date. We expect that this technique will be able to extend many other standard paleomagnetic techniques to previously inaccessible microscale samples.

Use of Kelvin probe force microscopy for identification of CVD grown graphene flakes on copper foil

Science.gov (United States)

Kumar, Rakesh; Mehta, B. R.; Kanjilal, D.

2017-05-01

Graphene flakes have been grown by chemical vapour deposition (CVD) method on Cu foils. The obtained graphene flakes have been characterized by optical microscopy, field emission scanning electron microscopy, Kelvin probe force microscopy (KPFM) and Raman spectroscopy. The graphene flakes grown on Cu foil comprise mainly single layer graphene and confirm that the nucleation for graphene growth starts very quickly. Moreover, KPFM has been found to be a valuable technique to differentiate between covered and uncovered portion of Cu foil by graphene flakes deposited for shorter duration. The results show that KPFM can be a very useful technique in understanding the mechanism of graphene growth.

Selective plane illumination microscopy (SPIM) with time-domain fluorescence lifetime imaging microscopy (FLIM) for volumetric measurement of cleared mouse brain samples

Science.gov (United States)

Funane, Tsukasa; Hou, Steven S.; Zoltowska, Katarzyna Marta; van Veluw, Susanne J.; Berezovska, Oksana; Kumar, Anand T. N.; Bacskai, Brian J.

2018-05-01

We have developed an imaging technique which combines selective plane illumination microscopy with time-domain fluorescence lifetime imaging microscopy (SPIM-FLIM) for three-dimensional volumetric imaging of cleared mouse brains with micro- to mesoscopic resolution. The main features of the microscope include a wavelength-adjustable pulsed laser source (Ti:sapphire) (near-infrared) laser, a BiBO frequency-doubling photonic crystal, a liquid chamber, an electrically focus-tunable lens, a cuvette based sample holder, and an air (dry) objective lens. The performance of the system was evaluated with a lifetime reference dye and micro-bead phantom measurements. Intensity and lifetime maps of three-dimensional human embryonic kidney (HEK) cell culture samples and cleared mouse brain samples expressing green fluorescent protein (GFP) (donor only) and green and red fluorescent protein [positive Förster (fluorescence) resonance energy transfer] were acquired. The results show that the SPIM-FLIM system can be used for sample sizes ranging from single cells to whole mouse organs and can serve as a powerful tool for medical and biological research.

Parallel detection experiment of fluorescence confocal microscopy using DMD.

Science.gov (United States)

Wang, Qingqing; Zheng, Jihong; Wang, Kangni; Gui, Kun; Guo, Hanming; Zhuang, Songlin

2016-05-01

Parallel detection of fluorescence confocal microscopy (PDFCM) system based on Digital Micromirror Device (DMD) is reported in this paper in order to realize simultaneous multi-channel imaging and improve detection speed. DMD is added into PDFCM system, working to take replace of the single traditional pinhole in the confocal system, which divides the laser source into multiple excitation beams. The PDFCM imaging system based on DMD is experimentally set up. The multi-channel image of fluorescence signal of potato cells sample is detected by parallel lateral scanning in order to verify the feasibility of introducing the DMD into fluorescence confocal microscope. In addition, for the purpose of characterizing the microscope, the depth response curve is also acquired. The experimental result shows that in contrast to conventional microscopy, the DMD-based PDFCM system has higher axial resolution and faster detection speed, which may bring some potential benefits in the biology and medicine analysis. SCANNING 38:234-239, 2016. © 2015 Wiley Periodicals, Inc. © Wiley Periodicals, Inc.

Scanning electron microscopy physics of image formation and microanalysis

CERN Document Server

Reimer, Ludwig

1985-01-01

The aim of this book is to outline the physics of image formation, electron­ specimen interactions, imaging modes, the interpretation of micrographs and the use of quantitative modes "in scanning electron microscopy (SEM). lt forms a counterpart to Transmission Electron Microscopy (Vol. 36 of this Springer Series in Optical Sciences) . The book evolved from lectures delivered at the University of Münster and from a German text entitled Raster-Elektronenmikroskopie (Springer-Verlag), published in collaboration with my colleague Gerhard Pfefferkorn. In the introductory chapter, the principles of the SEM and of electron­ specimen interactions are described, the most important imaging modes and their associated contrast are summarized, and general aspects of eiemental analysis by x-ray and Auger electron emission are discussed. The electron gun and electron optics are discussed in Chap. 2 in order to show how an electron probe of small diameter can be formed, how the elec­ tron beam can be blanked at high fre...

Characterization and quantitative determination of calcium aluminate clinker phases through reflected light microscopy

International Nuclear Information System (INIS)

Marciano Junior, E.; Cunha Munhoz, F.A. da; Splettstoser Junior, J.; Placido, W.F.

1989-01-01

The identification and quantitative determination of phases in calcium aluminate clinker is of great importance to the producer, as it enables a better understanding of the cement and concrete properties, specially those concerning setting time and compressive strenght. Polished sections of three electrofused clinkers, one experimental and two industrial, were used to select the most suitable etchings in order to identify by microscopy the main phases (Ca, CA 2 , C 2 AS, C 12 A 7 , α-Al 2 O 3 ). Quantitative phases determinations by reflected light microscopy showed good results when compared to X-ray diffractometry measurements [pt

Single spin stochastic optical reconstruction microscopy

OpenAIRE

Pfender, Matthias; Aslam, Nabeel; Waldherr, Gerald; Wrachtrup, Jörg

2014-01-01

We experimentally demonstrate precision addressing of single quantum emitters by combined optical microscopy and spin resonance techniques. To this end we utilize nitrogen-vacancy (NV) color centers in diamond confined within a few ten nanometers as individually resolvable quantum systems. By developing a stochastic optical reconstruction microscopy (STORM) technique for NV centers we are able to simultaneously perform sub diffraction-limit imaging and optically detected spin resonance (ODMR)...

Reconstruction of Undersampled Atomic Force Microscopy Images

DEFF Research Database (Denmark)

Jensen, Tobias Lindstrøm; Arildsen, Thomas; Østergaard, Jan

2013-01-01

Atomic force microscopy (AFM) is one of the most advanced tools for high-resolution imaging and manipulation of nanoscale matter. Unfortunately, standard AFM imaging requires a timescale on the order of seconds to minutes to acquire an image which makes it complicated to observe dynamic processes....... Moreover, it is often required to take several images before a relevant observation region is identified. In this paper we show how to significantly reduce the image acquisition time by undersampling. The reconstruction of an undersampled AFM image can be viewed as an inpainting, interpolating problem...... should be reconstructed using interpolation....

Nanoscale surface characterization using laser interference microscopy

Science.gov (United States)

Ignatyev, Pavel S.; Skrynnik, Andrey A.; Melnik, Yury A.

2018-03-01

Nanoscale surface characterization is one of the most significant parts of modern materials development and application. The modern microscopes are expensive and complicated tools, and its use for industrial tasks is limited due to laborious sample preparation, measurement procedures, and low operation speed. The laser modulation interference microscopy method (MIM) for real-time quantitative and qualitative analysis of glass, metals, ceramics, and various coatings has a spatial resolution of 0.1 nm for vertical and up to 100 nm for lateral. It is proposed as an alternative to traditional scanning electron microscopy (SEM) and atomic force microscopy (AFM) methods. It is demonstrated that in the cases of roughness metrology for super smooth (Ra >1 nm) surfaces the application of a laser interference microscopy techniques is more optimal than conventional SEM and AFM. The comparison of semiconductor test structure for lateral dimensions measurements obtained with SEM and AFM and white light interferometer also demonstrates the advantages of MIM technique.

Imaging of human differentiated 3D neural aggregates using light sheet fluorescence microscopy

Science.gov (United States)

Gualda, Emilio J.; Simão, Daniel; Pinto, Catarina; Alves, Paula M.; Brito, Catarina

2014-01-01

The development of three dimensional (3D) cell cultures represents a big step for the better understanding of cell behavior and disease in a more natural like environment, providing not only single but multiple cell type interactions in a complex 3D matrix, highly resembling physiological conditions. Light sheet fluorescence microscopy (LSFM) is becoming an excellent tool for fast imaging of such 3D biological structures. We demonstrate the potential of this technique for the imaging of human differentiated 3D neural aggregates in fixed and live samples, namely calcium imaging and cell death processes, showing the power of imaging modality compared with traditional microscopy. The combination of light sheet microscopy and 3D neural cultures will open the door to more challenging experiments involving drug testing at large scale as well as a better understanding of relevant biological processes in a more realistic environment. PMID:25161607

Imaging of human differentiated 3D neural aggregates using light sheet fluorescence microscopy

Directory of Open Access Journals (Sweden)

Emilio J Gualda

2014-08-01

Full Text Available The development of three dimensional cell cultures represents a big step for the better understanding of cell behavior and disease in a more natural like environment, providing not only single but multiple cell type interactions in a complex three dimensional matrix, highly resembling physiological conditions. Light sheet fluorescence microscopy is becoming an excellent tool for fast imaging of such three-dimensional biological structures. We demonstrate the potential of this technique for the imaging of human differentiated 3D neural aggregates in fixed and live samples, namely calcium imaging and cell death processes, showing the power of imaging modality compared with traditional microscopy. The combination of light sheet microscopy and 3D neural cultures will open the door to more challenging experiments involving drug testing at large scale as well as a better understanding of relevant biological processes in a more realistic environment.

Atomic force microscopy imaging to measure precipitate volume fraction in nickel-based superalloys

International Nuclear Information System (INIS)

Bourhettar, A.; Troyon, M.; Hazotte, A.

1995-01-01

In nickel-based superalloys, quantitative analysis of scanning electron microscopy images fails in providing accurate microstructural data, whereas more efficient techniques are very time-consuming. As an alternative approach, the authors propose to perform quantitative analysis of atomic force microscopy images of polished/etched surfaces (quantitative microprofilometry). This permits the measurement of microstructural parameters and the depth of etching, which is the main source of measurement bias. Thus, nonbiased estimations can be obtained by extrapolation of the measurements up to zero etching depth. In this article, the authors used this approach to estimate the volume fraction of γ' precipitates in a nickel-based superalloy single crystal. Atomic force microscopy images of samples etched for different times show definition, homogeneity, and contrast high enough to perform image analysis. The result after extrapolation is in very good agreement with volume fraction values available from published reports

On the formation of lipid droplets in human adipocytes: the organization of the perilipin-vimentin cortex.

Directory of Open Access Journals (Sweden)

Hans Heid

Full Text Available We report on the heterogeneity and diversity of lipid droplets (LDs in early stages of adipogenesis by elucidating the cell and molecular biology of amphiphilic and cytoskeletal proteins regulating and stabilizing the generation of LDs in human adipose cells. A plethora of distinct and differently sized LDs was detected by a brief application of adipocyte differentiation medium and additional short treatment with oleic acid. Using these cells and highly specific antibodies for LD-binding proteins of the perilipin (PLIN family, we could distinguish between endogenously derived LDs (endogenous LDs positive for perilipin from exogenously induced LDs (exogenous LDs positive for adipophilin, TIP47 and S3-12. Having optimized these stimulation conditions, we used early adipogenic differentiation stages to investigate small-sized LDs and concentrated on LD-protein associations with the intermediate-sized filament (IF vimentin. This IF protein was described earlier to surround lipid globules, showing spherical, cage-like structures. Consequently - by biochemical methods, by immunofluorescence microscopy and by electron- and immunoelectron microscopy - various stages of emerging lipid globules were revealed with perilipin as linking protein between LDs and vimentin. For this LD-PLIN-Vimentin connection, a model is now proposed, suggesting an interaction of proteins via opposed charged amino acid domains respectively. In addition, multiple sheaths of smooth endoplasmic reticulum cisternae surrounding concentrically nascent LDs are shown. Based on our comprehensive localization studies we present and discuss a novel pathway for the LD formation.

[Contribution of confocal microscopy and anterior chamber OCT to the study of corneal endothelial pathologies].

Science.gov (United States)

Fayol, N; Labbé, A; Dupont-Monod, S; Dupas, B; Baudouin, C

2007-04-01

To describe the appearance of various endothelial diseases with in vivo confocal microscopy and anterior chamber optical coherence tomography (AC OCT). In this study, ten patients with five different corneal endothelial pathologies were evaluated. Three patients had cornea guttata, three had corneal endothelial precipitates, two had irido-corneo-endothelial (ICE) syndrome, one had endothelial folds, and one had breaks in the Descemet membrane. All patients had bilateral ophthalmologic examinations, in vivo confocal microscopy, and AC OCT analysis. In cases of cornea guttata, AC OCT showed a finely embossed line corresponding to the empty intercellular cavities found with in vivo confocal microscopy. Corneal endothelium precipitates had the aspect of round formations suspended with the endothelium. Iris atrophy and irido-corneal synechiae resulting from ICE syndrome were precisely visualized with the AC OCT. High-resolution images of the anterior segment could be obtained using the AC OCT. Associated with in vivo confocal microscopy, these two new imaging techniques provide a precise evaluation of endothelial pathologies.

3rd International Multidisciplinary Microscopy and Microanalysis Congress

CERN Document Server

Oral, Zehra

2017-01-01

The 3rd International Multidisciplinary Microscopy Congress (InterM2015), held from 19 to 23 October 2015, focused on the latest developments concerning applications of microscopy in the biological, physical and chemical sciences at all dimensional scales, advances in instrumentation, techniques in and educational materials on microscopy. These proceedings gather 17 peer-reviewed technical papers submitted by leading academic and research institutions from nine countries and representing some of the most cutting-edge research available.

Multimodal nonlinear microscopy: A powerful label-free method for supporting standard diagnostics on biological tissues

Directory of Open Access Journals (Sweden)

Riccardo Cicchi

2014-09-01

Full Text Available The large use of nonlinear laser scanning microscopy in the past decade paved the way for potential clinical application of this imaging technique. Modern nonlinear microscopy techniques offer promising label-free solutions to improve diagnostic performances on tissues. In particular, the combination of multiple nonlinear imaging techniques in the same microscope allows integrating morphological with functional information in a morpho-functional scheme. Such approach provides a high-resolution label-free alternative to both histological and immunohistochemical examination of tissues and is becoming increasingly popular among the clinical community. Nevertheless, several technical improvements, including automatic scanning and image analysis, are required before the technique represents a standard diagnostic method. In this review paper, we highlight the capabilities of multimodal nonlinear microscopy for tissue imaging, by providing various examples on colon, arterial and skin tissues. The comparison between images acquired using multimodal nonlinear microscopy and histology shows a good agreement between the two methods. The results demonstrate that multimodal nonlinear microscopy is a powerful label-free alternative to standard histopathological methods and has the potential to find a stable place in the clinical setting in the near future.

New directions in point-contact spectroscopy based on scanning tunneling microscopy techniques (Review Article)

International Nuclear Information System (INIS)

Tartaglini, E.; Verhagen, T.G.A.; Galli, F.; Trouwborst, M.L.; Aarts, J.; Van-Ruitebbeek, J.M.; Muller, R.; Shiota, T.

2013-01-01

Igor Yanson showed 38 years ago for the first time a point-contact measurement where he probed the energy resolved spectroscopy of the electronic scattering inside the metal. Since this first measurement, the pointcontact spectroscopy (PCS) technique improved enormously. The application of the scanning probe microscopy (SPM) techniques in the late 1980s allowed achieving contacts with a diameter of a single atom. With the introduction of the mechanically controlled break junction technique, even spectroscopy on freely suspended chains of atoms could be performed. In this paper, we briefly review the current developments of PCS and show recent experiments in advanced scanning PCS based on SPM techniques. We describe some results obtained with both needle-anvil type of point contacts and scanning tunneling microscopy (STM). We also show our first attempt to lift up with a STM a chain of single gold atoms from a Au(110) surface.

Multiphoton microscopy imaging of developing tooth germs

Directory of Open Access Journals (Sweden)

Pei-Yu Pan

2014-01-01

Conclusion: In this study, a novel multiphoton microscopy database of images from developing tooth germs in mice was set up. We confirmed that multiphoton laser microscopy is a powerful tool for investigating the development of tooth germ and is worthy for further application in the study of tooth regeneration.

Magnetic force microscopy : Quantitative issues in biomaterials

NARCIS (Netherlands)

Passeri, D.; Dong, C.; Reggente, M.; Angeloni, L.; Barteri, M.; Scaramuzzo, F.A.; De Angelis, F.; Marinelli, F.; Antonelli, F.; Rinaldi, F.; Marianecci, C.; Carafa, M.; Sorbo, A.; Sordi, D.; Arends, I.W.C.E.; Rossi, M.

2014-01-01

Magnetic force microscopy (MFM) is an atomic force microscopy (AFM) based technique in which an AFM tip with a magnetic coating is used to probe local magnetic fields with the typical AFM spatial resolution, thus allowing one to acquire images reflecting the local magnetic properties of the samples

Resonant Scanning with Large Field of View Reduces Photobleaching and Enhances Fluorescence Yield in STED Microscopy.

Science.gov (United States)

Wu, Yong; Wu, Xundong; Lu, Rong; Zhang, Jin; Toro, Ligia; Stefani, Enrico

2015-10-01

Photobleaching is a major limitation of superresolution Stimulated Depletion Emission (STED) microscopy. Fast scanning has long been considered an effective means to reduce photobleaching in fluorescence microscopy, but a careful quantitative study of this issue is missing. In this paper, we show that the photobleaching rate in STED microscopy can be slowed down and the fluorescence yield be enhanced by scanning with high speed, enabled by using large field of view in a custom-built resonant-scanning STED microscope. The effect of scanning speed on photobleaching and fluorescence yield is more remarkable at higher levels of depletion laser irradiance, and virtually disappears in conventional confocal microscopy. With ≥6 GW∙cm(-2) depletion irradiance, we were able to extend the fluorophore survival time of Atto 647N and Abberior STAR 635P by ~80% with 8-fold wider field of view. We confirm that STED Photobleaching is primarily caused by the depletion light acting upon the excited fluorophores. Experimental data agree with a theoretical model. Our results encourage further increasing the linear scanning speed for photobleaching reduction in STED microscopy.

Studies on silica deposition in sugarcane (Saccharum spp. ) using scanning electron microscopy, energy-dispersive X-ray analysis, neutron activation analysis, and light microscopy

Energy Technology Data Exchange (ETDEWEB)

Kaufman, P B; Takeoka, Y; Carlson, T J; Bigelow, W C; Jones, J D; Moore, P H; Ghosheh, N S [Michigan Univ., Ann Arbor (USA)

1979-06-01

Marked differences in silicon content in internodes of two sugarcane cultivars as revealed by neutron activation analysis, were closely correlated with number of silica cells per unit area in the epidermal system of the internodes of the two cultivars, as indicated by scanning electron microscopy and X-ray analysis. Light microscopy of epidermal peels showed that silica cells are capable of transmitting significantly more light through themselves than do other types of adjacent epidermal cells. This could be of great significance to total amount of carbon fixed by photosynthesizing mesophyll cells in leaves and cortical cells in internodes below the epidermis, especially in sugarcane cultivars with high densities of silica cells in their shoot epidermal systems. This has led to propose a window hypothesis, which indicates that silica cells in sugarcane, and in other grasses, act like windows in the epidermal system, allowing more light to be transmitted to photosynthetic tissue below than would occur if silica cells were absent.

Scanning microscopies of superconductors at very low temperatures

International Nuclear Information System (INIS)

Crespo, V.; Maldonado, A.; Galvis, J.A.; Kulkarni, P.; Guillamon, I.; Rodrigo, J.G.; Suderow, H.; Vieira, S.; Banerjee, S.; Rodiere, P.

2012-01-01

We discuss basics of Scanning Tunneling Microscopy and Spectroscopy (STM/S) of the superconducting state with normal and superconducting tips. We present a new method to measure the local variations in the Andreev reflection amplitude between a superconducting tip and the sample. This method is termed Scanning Andreev Reflection Spectroscopy (SAS). We also briefly discuss vortex imaging with STM/S under an applied current through the sample, and show the vortex lattice as a function of the angle between the magnetic field and sample’s surface.

New directions in scanning-tunneling microscopy

International Nuclear Information System (INIS)

Ferrell, T.L.; Warmack, R.J.; Reddick, R.C.

1989-01-01

The tunneling of electrons in scanning-tunneling microscopy (STM) has permitted imaging of the electronic distribution about individual atoms on surfaces. The need for use of conducting surfaces in STM limits its applicability, and new forms of scanning microscopy have emerged as a result of interest in poorly conducting samples. Atomic force microscopy has demonstrated that the force between a surface and a probe tip can be used to image selected materials. Now being developed are magnetic probe STM's and photon tunneling microscopes in which the probe is a sharpened optical fiber. Also of great interest presently is the measurement of differential conductance of surfaces using electron STM's. This method supplies spectral information and contrast enhancement in images. At present there remains much theoretical work to be carried out in order to better characterize related data on inelastic electron tunneling, and valuable insight may be gained from data being gathered on the local work function of materials. As matters stand today, the key problems lie in determining tip and contamination effects, preparation of samples, and understanding conductivity mechanisms in very thin materials on conducting substrates. Resolution of these problems and introduction of new forms of scanning microscopy may permit novel and important applications in biology as well as surface science

Investigation of porous asphalt microstructure using optical and electron microscopy.

Science.gov (United States)

Poulikakos, L D; Partl, M N

2010-11-01

Direct observations of porous asphalt concrete samples in their natural state using optical and electron microscopy techniques led to useful information regarding the microstructure of two mixes and indicated a relationship between microstructure and in situ performance. This paper presents evidence that suboptimal microstructure can lead to premature failure thus making a first step in defining well or suboptimal performing pavements with a bottom-up approach (microstructure). Laboratory and field compaction produce different samples in terms of the microstructure. Laboratory compaction using the gyratory method has produced more microcracks in mineral aggregates after the binder had cooled. Well-performing mixes used polymer-modified binders, had a more homogeneous void structure with fewer elongated voids and better interlocking of the aggregates. Furthermore, well-performing mixes showed better distribution of the mastic and better coverage of the aggregates with bitumen. Low vacuum scanning electron microscopy showed that styrene butadiene styrene polymer modification in binder exists in the form of discontinuous globules and not continuous networks. A reduction in the polymer phase was observed as a result of aging and in-service use. © 2010 The Authors Journal compilation © 2010 The Royal Microscopical Society.

Microscopy studies on pronton exchange membrane fuel cell electrodes with different ionomer contents

DEFF Research Database (Denmark)

Ma, Shuang; Solterbeck, Claus Henning; Odgaard, Madeleine

2009-01-01

of the electrode was well displayed in the topography and phase images. The particle and pore size (Z) distributions showed the most frequent values at 30-40 nm and 20-30 nm, respectively. The particle size corresponds to the size of the carbon support for the platinum catalyst. Catalyst agglomeration was observed......Proton Exchange Membrane (PEM) fuel cell electrodes with different ionomer contents were studied with various microscopic techniques. The morphology and surface potential were examined by Atomic Force Microscopy (AFM) and Kelvin Probe Microscopy (KPM), respectively. The particulate nature...... in high ionomer content electrodes. The surface potential images showed distinct difference to the topography images. The overall grain size was seen to increase, the pore volume to decrease, the surface roughness to decrease, and the surface potential variation to increase with the increase of ionomer...

Second harmonic generation microscopy differentiates collagen type I and type III in COPD

Science.gov (United States)

Suzuki, Masaru; Kayra, Damian; Elliott, W. Mark; Hogg, James C.; Abraham, Thomas

2012-03-01

The structural remodeling of extracellular matrix proteins in peripheral lung region is an important feature in chronic obstructive pulmonary disease (COPD). Multiphoton microscopy is capable of inducing specific second harmonic generation (SHG) signal from non-centrosymmetric structural proteins such as fibrillar collagens. In this study, SHG microscopy was used to examine structural remodeling of the fibrillar collagens in human lungs undergoing emphysematous destruction (n=2). The SHG signals originating from these diseased lung thin sections from base to apex (n=16) were captured simultaneously in both forward and backward directions. We found that the SHG images detected in the forward direction showed well-developed and well-structured thick collagen fibers while the SHG images detected in the backward direction showed striking different morphological features which included the diffused pattern of forward detected structures plus other forms of collagen structures. Comparison of these images with the wellestablished immunohistochemical staining indicated that the structures detected in the forward direction are primarily the thick collagen type I fibers and the structures identified in the backward direction are diffusive structures of forward detected collagen type I plus collagen type III. In conclusion, we here demonstrate the feasibility of SHG microscopy in differentiating fibrillar collagen subtypes and understanding their remodeling in diseased lung tissues.

Enhancement of fluorescence confocal scanning microscopy lateral resolution by use of structured illumination

International Nuclear Information System (INIS)

Kim, Taejoong; Gweon, DaeGab; Lee, Jun-Hee

2009-01-01

Confocal microscopy is an optical imaging technique used to reconstruct three-dimensional images without physical sectioning. As with other optical microscopes, the lateral resolution of the confocal microscope cannot surpass the diffraction limit. This paper presents a novel imaging system, structured illumination confocal scanning microscopy (SICSM), that uses structured illumination to improve the lateral resolution of the confocal microscope. The SICSM can easily be implemented by introducing a structured illumination generating optics to conventional line-scanning fluorescence confocal microscopy. In this paper, we report our analysis of the lateral and axial resolutions of the SICSM by use of mathematical imaging theory. Numerical simulation results show that the lateral resolution of the SICSM is 1.43-fold better than that of the confocal microscope. In the axial direction, however, the resolution of the SICSM is ∼15% poorer than that of the confocal microscope. This deterioration arises because of a decrease in the axial cut-off frequency caused by the process of generating structured illumination. We propose the use of imaging conditions under which a compromise between the axial and lateral resolutions is chosen. Finally, we show simulated images of diversely shaped test objects to demonstrate the lateral and axial resolution performance of the SICSM

Ultrafast Science Opportunities with Electron Microscopy

Energy Technology Data Exchange (ETDEWEB)

DURR, HERMANN; Wang, X.J., ed.

2016-04-28

X-rays and electrons are two of the most fundamental probes of matter. When the Linac Coherent Light Source (LCLS), the world’s first x-ray free electron laser, began operation in 2009, it transformed ultrafast science with the ability to generate laser-like x-ray pulses from the manipulation of relativistic electron beams. This document describes a similar future transformation. In Transmission Electron Microscopy, ultrafast relativistic (MeV energy) electron pulses can achieve unsurpassed spatial and temporal resolution. Ultrafast temporal resolution will be the next frontier in electron microscopy and can ideally complement ultrafast x-ray science done with free electron lasers. This document describes the Grand Challenge science opportunities in chemistry, material science, physics and biology that arise from an MeV ultrafast electron diffraction & microscopy facility, especially when coupled with linac-based intense THz and X-ray pump capabilities.

The role of helium ion microscopy in the characterisation of complex three-dimensional nanostructures

International Nuclear Information System (INIS)

Rodenburg, C.; Liu, X.; Jepson, M.A.E.; Zhou, Z.; Rainforth, W.M.; Rodenburg, J.M.

2010-01-01

This work addresses two major issues relating to Helium Ion Microscopy (HeIM). First we show that HeIM is capable of solving the interpretation difficulties that arise when complex three-dimensional structures are imaged using traditional high lateral resolution techniques which are transmission based, such as scanning transmission electron microscopy (STEM). Secondly we use a nano-composite coating consisting of amorphous carbon embedded in chromium rich matrix to estimate the mean escape depth for amorphous carbon for secondary electrons generated by helium ion impact as a measure of HeIM depth resolution.

Human tooth pulp anatomy visualization by 3D magnetic resonance microscopy

International Nuclear Information System (INIS)

Sustercic, Dusan; Sersa, Igor

2012-01-01

Precise assessment of dental pulp anatomy is of an extreme importance for a successful endodontic treatment. As standard radiographs of teeth provide very limited information on dental pulp anatomy, more capable methods are highly appreciated. One of these is 3D magnetic resonance (MR) microscopy of which diagnostic capabilities in terms of a better dental pulp anatomy assessment were evaluated in the study. Twenty extracted human teeth were scanned on a 2.35 T MRI system for MR microscopy using the 3D spin-echo method that enabled image acquisition with isotropic resolution of 100 μm. The 3D images were then post processed by ImageJ program (NIH) to obtain advanced volume rendered views of dental pulps. MR microscopy at 2.35 T provided accurate data on dental pulp anatomy in vitro. The data were presented as a sequence of thin 2D slices through the pulp in various orientations or as volume rendered 3D images reconstructed form arbitrary view-points. Sequential 2D images enabled only an approximate assessment of the pulp, while volume rendered 3D images were more precise in visualization of pulp anatomy and clearly showed pulp diverticles, number of pulp canals and root canal anastomosis. This in vitro study demonstrated that MR microscopy could provide very accurate 3D visualization of dental pulp anatomy. A possible future application of the method in vivo may be of a great importance for the endodontic treatment

Resolution improvement by nonconfocal theta microscopy.

Science.gov (United States)

Lindek, S; Stelzer, E H

1999-11-01

We present a novel scanning fluorescence microscopy technique, nonconfocal theta microscopy (NCTM), that provides almost isotropic resolution. In NCTM, multiphoton absorption from two orthogonal illumination directions is used to induce fluorescence emission. Therefore the point-spread function of the microscope is described by the product of illumination point-spread functions with reduced spatial overlap, which provides the resolution improvement and the more isotropic observation volume. We discuss the technical details of this new method.

Superresolution Microscopy of the Nuclear Envelope and Associated Proteins.

Science.gov (United States)

Xie, Wei; Horn, Henning F; Wright, Graham D

2016-01-01

Superresolution microscopy is undoubtedly one of the most exciting technologies since the invention of the optical microscope. Capable of nanometer-scale resolution to surpass the diffraction limit and coupled with the versatile labeling techniques available, it is revolutionizing the study of cell biology. Our understanding of the nucleus, the genetic and architectural center of the cell, has gained great advancements through the application of various superresolution microscopy techniques. This chapter describes detailed procedures of multichannel superresolution imaging of the mammalian nucleus, using structured illumination microscopy and single-molecule localization microscopy.

Fast time-resolved electrostatic force microscopy: Achieving sub-cycle time resolution

Energy Technology Data Exchange (ETDEWEB)

Karatay, Durmus U.; Harrison, Jeffrey S.; Glaz, Micah S.; Giridharagopal, Rajiv; Ginger, David S., E-mail: ginger@chem.washington.edu [Department of Chemistry, University of Washington, Seattle, Washington 98195 (United States)

2016-05-15

The ability to measure microsecond- and nanosecond-scale local dynamics below the diffraction limit with widely available atomic force microscopy hardware would enable new scientific studies in fields ranging from biology to semiconductor physics. However, commercially available scanning-probe instruments typically offer the ability to measure dynamics only on time scales of milliseconds to seconds. Here, we describe in detail the implementation of fast time-resolved electrostatic force microscopy using an oscillating cantilever as a means to measure fast local dynamics following a perturbation to a sample. We show how the phase of the oscillating cantilever relative to the perturbation event is critical to achieving reliable sub-cycle time resolution. We explore how noise affects the achievable time resolution and present empirical guidelines for reducing noise and optimizing experimental parameters. Specifically, we show that reducing the noise on the cantilever by using photothermal excitation instead of piezoacoustic excitation further improves time resolution. We demonstrate the discrimination of signal rise times with time constants as fast as 10 ns, and simultaneous data acquisition and analysis for dramatically improved image acquisition times.

Effect of SP-C on surface potential distribution in pulmonary surfactant: Atomic force microscopy and Kelvin probe force microscopy study

International Nuclear Information System (INIS)

Hane, Francis; Moores, Brad; Amrein, Matthias; Leonenko, Zoya

2009-01-01

The air-lung interface is covered by a molecular film of pulmonary surfactant (PS). The major function of the film is to reduce the surface tension of the lung's air-liquid interface, providing stability to the alveolar structure and reducing the work of breathing. Earlier we have shown that function of bovine lipid extract surfactant (BLES) is related to the specific molecular architecture of surfactant films. Defined molecular arrangement of the lipids and proteins of the surfactant film also give rise to a local highly variable electrical surface potential of the interface. In this work we investigated a simple model of artificial lung surfactant consisting of DPPC, eggPG, and surfactant protein C (SP-C). Effects of surface compression and the presence of SP-C on the monolayer structure and surface potential distribution were investigated using atomic force microscopy (AFM) and Kelvin probe force microscopy (KPFM). We show that topography and locally variable surface potential of DPPC-eggPG lipid mixture are similar to those of pulmonary surfactant BLES in the presence of SP-C and differ in surface potential when SP-C is absent.

Boundary segmentation for fluorescence microscopy using steerable filters

Science.gov (United States)

Ho, David Joon; Salama, Paul; Dunn, Kenneth W.; Delp, Edward J.

2017-02-01

Fluorescence microscopy is used to image multiple subcellular structures in living cells which are not readily observed using conventional optical microscopy. Moreover, two-photon microscopy is widely used to image structures deeper in tissue. Recent advancement in fluorescence microscopy has enabled the generation of large data sets of images at different depths, times, and spectral channels. Thus, automatic object segmentation is necessary since manual segmentation would be inefficient and biased. However, automatic segmentation is still a challenging problem as regions of interest may not have well defined boundaries as well as non-uniform pixel intensities. This paper describes a method for segmenting tubular structures in fluorescence microscopy images of rat kidney and liver samples using adaptive histogram equalization, foreground/background segmentation, steerable filters to capture directional tendencies, and connected-component analysis. The results from several data sets demonstrate that our method can segment tubular boundaries successfully. Moreover, our method has better performance when compared to other popular image segmentation methods when using ground truth data obtained via manual segmentation.

Platinum replica electron microscopy: Imaging the cytoskeleton globally and locally.

Science.gov (United States)

Svitkina, Tatyana M

2017-05-01

Structural studies reveal how smaller components of a system work together as a whole. However, combining high resolution of details with full coverage of the whole is challenging. In cell biology, light microscopy can image many cells in their entirety, but at a lower resolution, whereas electron microscopy affords very high resolution, but usually at the expense of the sample size and coverage. Structural analyses of the cytoskeleton are especially demanding, because cytoskeletal networks are unresolvable by light microscopy due to their density and intricacy, whereas their proper preservation is a challenge for electron microscopy. Platinum replica electron microscopy can uniquely bridge the gap between the "comfort zones" of light and electron microscopy by allowing high resolution imaging of the cytoskeleton throughout the entire cell and in many cells in the population. This review describes the principles and applications of platinum replica electron microscopy for studies of the cytoskeleton. Copyright © 2017 Elsevier Ltd. All rights reserved.

Ultrasonic force microscopy: detection and imaging of ultra-thin molecular domains.

Science.gov (United States)

Dinelli, Franco; Albonetti, Cristiano; Kolosov, Oleg V

2011-03-01

The analysis of the formation of ultra-thin organic films is a very important issue. In fact, it is known that the properties of organic light emitting diodes and field effect transistors are strongly affected by the early growth stages. For instance, in the case of sexithiophene, the presence of domains made of molecules with the backbone parallel to the substrate surface has been indirectly evidenced by photoluminescence spectroscopy and confocal microscopy. On the contrary, conventional scanning force microscopy both in contact and intermittent contact modes have failed to detect such domains. In this paper, we show that Ultrasonic Force Microscopy (UFM), sensitive to nanomechanical properties, allows one to directly identify the structure of sub-monolayer thick films. Sexithiophene flat domains have been imaged for the first time with nanometer scale spatial resolution. A comparison with lateral force and intermittent contact modes has been carried out in order to explain the origins of the UFM contrast and its advantages. In particular, it indicates that UFM is highly suitable for investigations where high sensitivity to material properties, low specimen damage and high spatial resolution are required. Copyright © 2010 Elsevier B.V. All rights reserved.

Analysis of ancient pigments by Raman microscopy

International Nuclear Information System (INIS)

Zuo Jian; Xu Cunyi

1999-01-01

Raman microscopy can be applied for the spatial resolution, and non-destructive in situ analysis of inorganic pigments in pottery, manuscripts and paintings. Compared with other techniques, it is the best single technique for this purpose. An overview is presented of the applications of Raman microscopy in the analysis of ancient pigments

Phenol-ammonium sulfate microscopy method for diagnosis of pulmonary tuberculosis

Directory of Open Access Journals (Sweden)

Ritu Singhal

2013-01-01

Conclusion: Sputum microscopy with PhAS solution is a safe, reliable and inexpensive alternative for direct microscopy. This method can be conveniently applied for usage in microscopy centers with limited bio-safety facilities.

Electron microscopy characterization of Ni-Cr-B-Si-C laser deposited coatings.

Science.gov (United States)

Hemmati, I; Rao, J C; Ocelík, V; De Hosson, J Th M

2013-02-01

During laser deposition of Ni-Cr-B-Si-C alloys with high amounts of Cr and B, various microstructures and phases can be generated from the same chemical composition that results in heterogeneous properties in the clad layer. In this study, the microstructure and phase constitution of a high-alloy Ni-Cr-B-Si-C coating deposited by laser cladding were analyzed by a combination of several microscopy characterization techniques including scanning electron microscopy in secondary and backscatter imaging modes, energy dispersive spectroscopy (EDS), electron backscatter diffraction (EBSD), and transmission electron microscopy (TEM). The combination of EDS and EBSD allowed unequivocal identification of micron-sized precipitates as polycrystalline orthorhombic CrB, single crystal tetragonal Cr5B3, and single crystal hexagonal Cr7C3. In addition, TEM characterization showed various equilibrium and metastable Ni-B, Ni-Si, and Ni-Si-B eutectic products in the alloy matrix. The findings of this study can be used to explain the phase formation reactions and to tune the microstructure of Ni-Cr-B-Si-C coatings to obtain the desired properties.

Strand-like structures and the nonstructural proteins 5, 3 and 1 are present in the nucleus of mosquito cells infected with dengue virus.

Science.gov (United States)

Reyes-Ruiz, José M; Osuna-Ramos, Juan F; Cervantes-Salazar, Margot; Lagunes Guillen, Anel E; Chávez-Munguía, Bibiana; Salas-Benito, Juan S; Del Ángel, Rosa M

2018-02-01

Dengue virus (DENV) is an arbovirus, which replicates in the endoplasmic reticulum. Although replicative cycle takes place in the cytoplasm, some viral proteins such as NS5 and C are translocated to the nucleus during infection in mosquitoes and mammalian cells. To localized viral proteins in DENV-infected C6/36 cells, an immunofluorescence (IF) and immunoelectron microscopy (IEM) analysis were performed. Our results indicated that C, NS1, NS3 and NS5 proteins were found in the nucleus of DENV-infected C6/36 cells. Additionally, complex structures named strand-like structures (Ss) were observed in the nucleus of infected cells. Interestingly, the NS5 protein was located in these structures. Ss were absent in mock-infected cells, suggesting that DENV induces their formation in the nucleus of infected mosquito cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Aberrations and adaptive optics in super-resolution microscopy

Science.gov (United States)

Booth, Martin; Andrade, Débora; Burke, Daniel; Patton, Brian; Zurauskas, Mantas

2015-01-01

As one of the most powerful tools in the biological investigation of cellular structures and dynamic processes, fluorescence microscopy has undergone extraordinary developments in the past decades. The advent of super-resolution techniques has enabled fluorescence microscopy – or rather nanoscopy – to achieve nanoscale resolution in living specimens and unravelled the interior of cells with unprecedented detail. The methods employed in this expanding field of microscopy, however, are especially prone to the detrimental effects of optical aberrations. In this review, we discuss how super-resolution microscopy techniques based upon single-molecule switching, stimulated emission depletion and structured illumination each suffer from aberrations in different ways that are dependent upon intrinsic technical aspects. We discuss the use of adaptive optics as an effective means to overcome this problem. PMID:26124194

Diatom Valve Three-Dimensional Representation: A New Imaging Method Based on Combined Microscopies

Science.gov (United States)

Ferrara, Maria Antonietta; De Tommasi, Edoardo; Coppola, Giuseppe; De Stefano, Luca; Rea, Ilaria; Dardano, Principia

2016-01-01

The frustule of diatoms, unicellular microalgae, shows very interesting photonic features, generally related to its complicated and quasi-periodic micro- and nano-structure. In order to simulate light propagation inside and through this natural structure, it is important to develop three-dimensional (3D) models for synthetic replica with high spatial resolution. In this paper, we present a new method that generates images of microscopic diatoms with high definition, by merging scanning electron microscopy and digital holography microscopy or atomic force microscopy data. Starting from two digital images, both acquired separately with standard characterization procedures, a high spatial resolution (Δz = λ/20, Δx = Δy ≅ 100 nm, at least) 3D model of the object has been generated. Then, the two sets of data have been processed by matrix formalism, using an original mathematical algorithm implemented on a commercially available software. The developed methodology could be also of broad interest in the design and fabrication of micro-opto-electro-mechanical systems. PMID:27690008

Super-resolution microscopy reveals cell wall dynamics and peptidoglycan architecture in ovococcal bacteria.

Science.gov (United States)

Wheeler, Richard; Mesnage, Stéphane; Boneca, Ivo G; Hobbs, Jamie K; Foster, Simon J

2011-12-01

Cell morphology and viability in Eubacteria is dictated by the architecture of peptidoglycan, the major and essential structural component of the cell wall. Although the biochemical composition of peptidoglycan is well understood, how the peptidoglycan architecture can accommodate the dynamics of growth and division while maintaining cell shape remains largely unknown. Here, we elucidate the peptidoglycan architecture and dynamics of bacteria with ovoid cell shape (ovococci), which includes a number of important pathogens, by combining biochemical analyses with atomic force and super-resolution microscopies. Atomic force microscopy analysis showed preferential orientation of the peptidoglycan network parallel to the short axis of the cell, with distinct architectural features associated with septal and peripheral wall synthesis. Super-resolution three-dimensional structured illumination fluorescence microscopy was applied for the first time in bacteria to unravel the dynamics of peptidoglycan assembly in ovococci. The ovococci have a unique peptidoglycan architecture and growth mode not observed in other model organisms. © 2011 Blackwell Publishing Ltd.

Development of X-ray excitable luminescent probes for scanning X-ray microscopy

International Nuclear Information System (INIS)

Moronne, M.M.

1999-01-01

Transmission soft X-ray microscopy is now capable of achieving resolutions that are typically 5 times better than the best-visible light microscopes. With expected improvements in zone plate optics, an additional factor of two may be realized within the next few years. Despite the high resolution now available with X-ray microscopes and the high X-ray contrast provided by biological molecules in the soft X-ray region (λ=2-5 nm), molecular probes for localizing specific biological targets have been lacking. To circumvent this problem, X-ray excitable molecular probes are needed that can target unique biological features. In this paper we report our initial results on the development of lanthanide-based fluorescent probes for biological labeling. Using scanning luminescence X-ray microscopy (SLXM, Jacobsen et al., J. Microscopy 172 (1993) 121-129), we show that lanthanide organo-polychelate complexes are sufficiently bright and radiation resistant to be the basis of a new class of X-ray excitable molecular probes capable of providing at least a fivefold improvement in resolution over visible light microscopy. Lanthanide probes, able to bind 80-100 metal ions per molecule, were found to give strong luminescent signals with X-ray doses exceeding 10 8 Gy, and were used to label actin stress fibers and in vitro preparations of polymerized tubulin. (Copyright (c) 1999 Elsevier Science B.V., Amsterdam. All rights reserved.)

A novel self-sensing technique for tapping-mode atomic force microscopy

Energy Technology Data Exchange (ETDEWEB)

Ruppert, Michael G.; Moheimani, S. O. Reza [The University of Newcastle, University Drive, Callaghan NSW 2308 (Australia)

2013-12-15

This work proposes a novel self-sensing tapping-mode atomic force microscopy operation utilizing charge measurement. A microcantilever coated with a single piezoelectric layer is simultaneously used for actuation and deflection sensing. The cantilever can be batch fabricated with existing micro electro mechanical system processes. The setup enables the omission of the optical beam deflection technique which is commonly used to measure the cantilever oscillation amplitude. Due to the high amount of capacitive feedthrough in the measured charge signal, a feedforward control technique is employed to increase the dynamic range from less than 1 dB to approximately 35 dB. Experiments show that the conditioned charge signal achieves excellent signal-to-noise ratio and can therefore be used as a feedback signal for atomic force microscopy imaging.

Electrostatic force microscopy: imaging DNA and protein polarizations one by one

International Nuclear Information System (INIS)

Mikamo-Satoh, Eriko; Yamada, Fumihiko; Takagi, Akihiko; Matsumoto, Takuya; Kawai, Tomoji

2009-01-01

We present electrostatic force microscopy images of double-stranded DNA and transcription complex on an insulating mica substrate obtained with molecular resolution using a frequency-mode noncontact atomic force microscope. The electrostatic potential images show that both DNA and transcription complexes are polarized with an upward dipole moment. Potential differences of these molecules from the mica substrate enabled us to estimate dipole moments of isolated DNA and transcription complex in zero external field to be 0.027 D/base and 0.16 D/molecule, respectively. Scanning capacitance microscopy demonstrates characteristic contrast inversion between DNA and transcription complex images, indicating the difference in electric polarizability of these molecules. These findings indicate that the electrostatic properties of individual biological molecules can be imaged on an insulator substrate while retaining complex formation.

High-resolution electron microscopy

CERN Document Server

Spence, John C H

2013-01-01

This new fourth edition of the standard text on atomic-resolution transmission electron microscopy (TEM) retains previous material on the fundamentals of electron optics and aberration correction, linear imaging theory (including wave aberrations to fifth order) with partial coherence, and multiple-scattering theory. Also preserved are updated earlier sections on practical methods, with detailed step-by-step accounts of the procedures needed to obtain the highest quality images of atoms and molecules using a modern TEM or STEM electron microscope. Applications sections have been updated - these include the semiconductor industry, superconductor research, solid state chemistry and nanoscience, and metallurgy, mineralogy, condensed matter physics, materials science and material on cryo-electron microscopy for structural biology. New or expanded sections have been added on electron holography, aberration correction, field-emission guns, imaging filters, super-resolution methods, Ptychography, Ronchigrams, tomogr...

Characterization and interactome study of white spot syndrome virus envelope protein VP11.

Directory of Open Access Journals (Sweden)

Wang-Jing Liu

Full Text Available White spot syndrome virus (WSSV is a large enveloped virus. The WSSV viral particle consists of three structural layers that surround its core DNA: an outer envelope, a tegument and a nucleocapsid. Here we characterize the WSSV structural protein VP11 (WSSV394, GenBank accession number AF440570, and use an interactome approach to analyze the possible associations between this protein and an array of other WSSV and host proteins. Temporal transcription analysis showed that vp11 is an early gene. Western blot hybridization of the intact viral particles and fractionation of the viral components, and immunoelectron microscopy showed that VP11 is an envelope protein. Membrane topology software predicted VP11 to be a type of transmembrane protein with a highly hydrophobic transmembrane domain at its N-terminal. Based on an immunofluorescence assay performed on VP11-transfected Sf9 cells and a trypsin digestion analysis of the virion, we conclude that, contrary to topology software prediction, the C-terminal of this protein is in fact inside the virion. Yeast two-hybrid screening combined with co-immunoprecipitation assays found that VP11 directly interacted with at least 12 other WSSV structural proteins as well as itself. An oligomerization assay further showed that VP11 could form dimers. VP11 is also the first reported WSSV structural protein to interact with the major nucleocapsid protein VP664.

Multiphoton Microscopy for Ophthalmic Imaging

Directory of Open Access Journals (Sweden)

Emily A. Gibson

2011-01-01

Full Text Available We review multiphoton microscopy (MPM including two-photon autofluorescence (2PAF, second harmonic generation (SHG, third harmonic generation (THG, fluorescence lifetime (FLIM, and coherent anti-Stokes Raman Scattering (CARS with relevance to clinical applications in ophthalmology. The different imaging modalities are discussed highlighting the particular strength that each has for functional tissue imaging. MPM is compared with current clinical ophthalmological imaging techniques such as reflectance confocal microscopy, optical coherence tomography, and fluorescence imaging. In addition, we discuss the future prospects for MPM in disease detection and clinical monitoring of disease progression, understanding fundamental disease mechanisms, and real-time monitoring of drug delivery.

Illuminating Electron Microscopy of Photocatalysts

DEFF Research Database (Denmark)

Cavalca, Filippo

.1% of the surface of the planet with a device that converts solar energy into a useable form at 10% efficiency would give more than the present worldwide consumption of fossil energy. Photocatalysts are of fundamental interest for sustainable energy research because they provide a viable route for converting solar...... energy into chemical bonds. By means of Transmission Electron Microscopy (TEM) it is possible to gain insight in the fundamentals of their reaction mechanisms, chemical behaviour, structure and morphology before, during and after reaction using in situ investigations. In particular, the environmental TEM...... the microscope that allows electron microscopy under nonconventional TEM conditions and new kinds of in situ spectroscopy....

Performance of microscopy and ELISA for diagnosing Giardia duodenalis infection in different pediatric groups.

Science.gov (United States)

Silva, Renata K N R; Pacheco, Flávia T F; Martins, Adson S; Menezes, Joelma F; Costa-Ribeiro, Hugo; Ribeiro, Tereza C M; Mattos, Ângela P; Oliveira, Ricardo R; Soares, Neci M; Teixeira, Márcia C A

2016-12-01

Techniques for Giardia diagnosis based on microscopy are usually applied as routine laboratory testing; however, they typically exhibit low sensitivity. This study aimed to evaluate Giardia duodenalis and other intestinal parasitic infections in different pediatric groups, with an emphasis on the comparison of Giardia diagnostic techniques. Feces from 824 children from different groups (diarrheic, malnourished, with cancer and from day care) were examined by microscopy and ELISA for Giardia, Cryptosporidium sp. and Entamoeba histolytica coproantigen detection. Giardia-positive samples from day-care children, identified by either microscopy or ELISA, were further tested by PCR targeting of the β-giardin and Gdh genes. Statistically significant differences (Psp. in diarrheic and malnourished groups; infections by Entamoeba histolytica were found only in children with diarrhea. Considering positivity for Giardia by at least one method, ELISA was found to be more sensitive than microscopy (97% versus 55%). To examine discrepancies among the diagnostic methods, 71 Giardia-positive stool samples from day-care children were tested by PCR; of these, DNA was amplified from 51 samples (77.4%). Concordance of positivity between microscopy and ELISA was found for 48 samples, with 43 confirmed by PCR. Parasite DNA was amplified from eleven of the 20 Giardia samples (55%) identified only by ELISA. This study shows the higher sensitivity of ELISA over microscopy for Giardia diagnosis when a single sample is analyzed and emphasizes the need for methods based on coproantigen detection to identify this parasite in diarrheic fecal samples. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Parallel detecting super-resolution microscopy using correlation based image restoration

Science.gov (United States)

Yu, Zhongzhi; Liu, Shaocong; Zhu, Dazhao; Kuang, Cuifang; Liu, Xu

2017-12-01

A novel approach to achieve the image restoration is proposed in which each detector's relative position in the detector array is no longer a necessity. We can identify each detector's relative location by extracting a certain area from one of the detector's image and scanning it on other detectors' images. According to this location, we can generate the point spread functions (PSF) for each detector and perform deconvolution for image restoration. Equipped with this method, the microscope with discretionally designed detector array can be easily constructed without the concern of exact relative locations of detectors. The simulated results and experimental results show the total improvement in resolution with a factor of 1.7 compared to conventional confocal fluorescence microscopy. With the significant enhancement in resolution and easiness for application of this method, this novel method should have potential for a wide range of application in fluorescence microscopy based on parallel detecting.

Thermal diffuse scattering in transmission electron microscopy

Energy Technology Data Exchange (ETDEWEB)

Forbes, B.D.; D' Alfonso, A.J. [School of Physics, University of Melbourne, Parkville, Victoria 3010 (Australia); Findlay, S.D. [School of Physics, Monash University, Victoria 3800 (Australia); Van Dyck, D. [EMAT, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp (Belgium); LeBeau, J.M. [North Carolina State University, Raleigh, NC 27695-7907 (United States); Stemmer, S. [Materials Department, University of California, Santa Barbara, CA 93106-5050 (United States); Allen, L.J., E-mail: lja@unimelb.edu.au [School of Physics, University of Melbourne, Parkville, Victoria 3010 (Australia)

2011-12-15

In conventional transmission electron microscopy, thermal scattering significantly affects the image contrast. It has been suggested that not accounting for this correctly is the main cause of the Stobbs factor, the ubiquitous, large contrast mismatch found between theory and experiment. In the case where a hard aperture is applied, we show that previous conclusions drawn from work using bright field scanning transmission electron microscopy and invoking the principle of reciprocity are reliable in the presence of thermal scattering. In the aperture-free case it has been suggested that even the most sophisticated mathematical models for thermal diffuse scattering lack in their numerical implementation, specifically that there may be issues in sampling, including that of the contrast transfer function of the objective lens. We show that these concerns can be satisfactorily overcome with modest computing resources; thermal scattering can be modelled accurately enough for the purpose of making quantitative comparison between simulation and experiment. Spatial incoherence of the source is also investigated. Neglect or inadequate handling of thermal scattering in simulation can have an appreciable effect on the predicted contrast and can be a significant contribution to the Stobbs factor problem. -- Highlights: Black-Right-Pointing-Pointer We determine the numerical requirements for accurate simulation of TDS in CTEM. Black-Right-Pointing-Pointer TDS can be simulated to high precision using the Born-Oppenheimer model. Black-Right-Pointing-Pointer Such calculations establish the contribution of TDS to the Stobbs factor problem. Black-Right-Pointing-Pointer Treating spatial incoherence using envelope functions increases image contrast. Black-Right-Pointing-Pointer Rigorous treatment of spatial incoherence significantly reduces image contrast.

Label-free three-dimensional imaging of cell nucleus using third-harmonic generation microscopy

Energy Technology Data Exchange (ETDEWEB)

Lin, Jian; Zheng, Wei; Wang, Zi; Huang, Zhiwei, E-mail: biehzw@nus.edu.sg [Optical Bioimaging Laboratory, Department of Biomedical Engineering, Faculty of Engineering, National University of Singapore, Singapore 117576 (Singapore)

2014-09-08

We report the implementation of the combined third-harmonic generation (THG) and two-photon excited fluorescence (TPEF) microscopy for label-free three-dimensional (3-D) imaging of cell nucleus morphological changes in liver tissue. THG imaging shows regular spherical shapes of normal hepatocytes nuclei with inner chromatin structures while revealing the condensation of chromatins and nuclear fragmentations in hepatocytes of diseased liver tissue. Colocalized THG and TPEF imaging provides complementary information of cell nuclei and cytoplasm in tissue. This work suggests that 3-D THG microscopy has the potential for quantitative analysis of nuclear morphology in cells at a submicron-resolution without the need for DNA staining.

Label-free three-dimensional imaging of cell nucleus using third-harmonic generation microscopy

International Nuclear Information System (INIS)

Lin, Jian; Zheng, Wei; Wang, Zi; Huang, Zhiwei

2014-01-01

We report the implementation of the combined third-harmonic generation (THG) and two-photon excited fluorescence (TPEF) microscopy for label-free three-dimensional (3-D) imaging of cell nucleus morphological changes in liver tissue. THG imaging shows regular spherical shapes of normal hepatocytes nuclei with inner chromatin structures while revealing the condensation of chromatins and nuclear fragmentations in hepatocytes of diseased liver tissue. Colocalized THG and TPEF imaging provides complementary information of cell nuclei and cytoplasm in tissue. This work suggests that 3-D THG microscopy has the potential for quantitative analysis of nuclear morphology in cells at a submicron-resolution without the need for DNA staining.

Application of multiphoton microscopy in dermatological studies: A mini-review

Directory of Open Access Journals (Sweden)

Elijah Yew

2014-09-01

Full Text Available This review summarizes the historical and more recent developments of multiphoton microscopy, as applied to dermatology. Multiphoton microscopy offers several advantages over competing microscopy techniques: there is an inherent axial sectioning, penetration depths that compete well with confocal microscopy on account of the use of near-infrared light, and many two-photon contrast mechanisms, such as second-harmonic generation, have no analogue in one-photon microscopy. While the penetration depths of photons into tissue are typically limited on the order of hundreds of microns, this is of less concern in dermatology, as the skin is thin and readily accessible. As a result, multiphoton microscopy in dermatology has generated a great deal of interest, much of which is summarized here. The review covers the interaction of light and tissue, as well as the various considerations that must be made when designing an instrument. The state of multiphoton microscopy in imaging skin cancer and various other diseases is also discussed, along with the investigation of aging and regeneration phenomena, and finally, the use of multiphoton microscopy to analyze the transdermal transport of drugs, cosmetics and other agents is summarized. The review concludes with a look at potential future research directions, especially those that are necessary to push these techniques into widespread clinical acceptance.

Functional photoacoustic microscopy of pH.

Science.gov (United States)

Chatni, Muhammad Rameez; Yao, Junjie; Danielli, Amos; Favazza, Christopher P; Maslov, Konstantin I; Wang, Lihong V

2011-10-01

pH is a tightly regulated indicator of metabolic activity. In mammalian systems, an imbalance of pH regulation may result from or result in serious illness. In this paper, we report photoacoustic microscopy (PAM) of a commercially available pH-sensitive fluorescent dye (SNARF-5F carboxylic acid) in tissue phantoms. We demonstrated that PAM is capable of pH imaging in absolute values at tissue depths of up to 2.0 mm, greater than possible with other forms of optical microscopy.

Type 1 diabetes mellitus effects on dental enamel formation revealed by microscopy and microanalysis.

Science.gov (United States)

Silva, Bruna Larissa Lago; Medeiros, Danila Lima; Soares, Ana Prates; Line, Sérgio Roberto Peres; Pinto, Maria das Graças Farias; Soares, Telma de Jesus; do Espírito Santo, Alexandre Ribeiro

2018-03-01

Type 1 diabetes mellitus (T1DM) largely affects children, occurring therefore at the same period of deciduous and permanent teeth development. The aim of this work was to investigate birefringence and morphology of the secretory stage enamel organic extracellular matrix (EOECM), and structural and mechanical features of mature enamel from T1DM rats. Adult Wistar rats were maintained alive for a period of 56 days after the induction of experimental T1DM with a single dose of streptozotocin (60 mg/kg). After proper euthanasia of the animals, fixed upper incisors were accurately processed, and secretory stage EOECM and mature enamel were analyzed by transmitted polarizing and bright field light microscopies (TPLM and BFLM), energy-dispersive x-ray (EDX) analysis, scanning electron microscopy (SEM), and microhardness testing. Bright field light microscopies and transmitted polarizing light microscopies showed slight morphological changes in the secretory stage EOECM from diabetic rats, which also did not exhibit statistically significant alterations in birefringence brightness when compared to control animals (P > .05). EDX analysis showed that T1DM induced statistically significant little increases in the amount of calcium and phosphorus in outer mature enamel (P  .05). T1DM also caused important ultrastructural alterations in mature enamel as revealed by SEM and induced a statistically significant reduction of about 13.67% in its microhardness at 80 μm from dentin-enamel junction (P enamel development, leading to alterations in mature enamel ultrastructure and in its mechanical features. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Nonlinear adaptive optics: aberration correction in three photon fluorescence microscopy for mouse brain imaging

Science.gov (United States)

Sinefeld, David; Paudel, Hari P.; Wang, Tianyu; Wang, Mengran; Ouzounov, Dimitre G.; Bifano, Thomas G.; Xu, Chris

2017-02-01

Multiphoton fluorescence microscopy is a well-established technique for deep-tissue imaging with subcellular resolution. Three-photon microscopy (3PM) when combined with long wavelength excitation was shown to allow deeper imaging than two-photon microscopy (2PM) in biological tissues, such as mouse brain, because out-of-focus background light can be further reduced due to the higher order nonlinear excitation. As was demonstrated in 2PM systems, imaging depth and resolution can be improved by aberration correction using adaptive optics (AO) techniques which are based on shaping the scanning beam using a spatial light modulator (SLM). In this way, it is possible to compensate for tissue low order aberration and to some extent, to compensate for tissue scattering. Here, we present a 3PM AO microscopy system for brain imaging. Soliton self-frequency shift is used to create a femtosecond source at 1675 nm and a microelectromechanical (MEMS) SLM serves as the wavefront shaping device. We perturb the 1020 segment SLM using a modified nonlinear version of three-point phase shifting interferometry. The nonlinearity of the fluorescence signal used for feedback ensures that the signal is increasing when the spot size decreases, allowing compensation of phase errors in an iterative optimization process without direct phase measurement. We compare the performance for different orders of nonlinear feedback, showing an exponential growth in signal improvement as the nonlinear order increases. We demonstrate the impact of the method by applying the 3PM AO system for in-vivo mouse brain imaging, showing improvement in signal at 1-mm depth inside the brain.

Fluctuation microscopy: a probe of medium range order

International Nuclear Information System (INIS)

Treacy, M M J; Gibson, J M; Fan, L; Paterson, D J; McNulty, I

2005-01-01

Fluctuation microscopy is a hybrid diffraction-imaging technique that detects medium range order in amorphous materials by examining spatial fluctuations in coherent scattering. These fluctuations appear as speckle in images and diffraction patterns. The volume of material contributing to the speckle is determined by the point-spread function (the resolution) of the imaging optics and the sample thickness. The spatial periodicities being probed are related to the diffraction vector. Statistical analysis of the speckle allows the random and non-random (ordered) contributions to be discriminated. The image resolution that gives the maximum speckle contrast, as determined by the normalized variance of the image intensity, is determined by the characteristic length scale of the ordering. Because medium range ordering length scales can extend out to about the tenth coordination shell, fluctuation microscopy tends to be a low image resolution technique. This review presents the kinematical scattering theory underpinning fluctuation microscopy and a description of fluctuation electron microscopy as it has been employed in the transmission electron microscope for studying amorphous materials. Recent results using soft x-rays for studying nanoscale materials are also presented. We summarize outstanding issues and point to possible future directions for fluctuation microscopy as a technique

Application of Confocal Laser Scanning Microscopy in Biology and Medicine

OpenAIRE

I. A. Volkov; N. V. Frigo; L. F. Znamenskaya; O. R. Katunina

2014-01-01

Fluorescence confocal laser scanning microscopy and reflectance confocal laser scanning microscopy are up-to-date highend study methods. Confocal microscopy is used in cell biology and medicine. By using confocal microscopy, it is possible to study bioplasts and localization of protein molecules and other compounds relative to cell or tissue structures, and to monitor dynamic cell processes. Confocal microscopes enable layer-by-layer scanning of test items to create demonstrable 3D models. As...

Optofluidic time-stretch quantitative phase microscopy.

Science.gov (United States)

Guo, Baoshan; Lei, Cheng; Wu, Yi; Kobayashi, Hirofumi; Ito, Takuro; Yalikun, Yaxiaer; Lee, Sangwook; Isozaki, Akihiro; Li, Ming; Jiang, Yiyue; Yasumoto, Atsushi; Di Carlo, Dino; Tanaka, Yo; Yatomi, Yutaka; Ozeki, Yasuyuki; Goda, Keisuke

2018-03-01

Innovations in optical microscopy have opened new windows onto scientific research, industrial quality control, and medical practice over the last few decades. One of such innovations is optofluidic time-stretch quantitative phase microscopy - an emerging method for high-throughput quantitative phase imaging that builds on the interference between temporally stretched signal and reference pulses by using dispersive properties of light in both spatial and temporal domains in an interferometric configuration on a microfluidic platform. It achieves the continuous acquisition of both intensity and phase images with a high throughput of more than 10,000 particles or cells per second by overcoming speed limitations that exist in conventional quantitative phase imaging methods. Applications enabled by such capabilities are versatile and include characterization of cancer cells and microalgal cultures. In this paper, we review the principles and applications of optofluidic time-stretch quantitative phase microscopy and discuss its future perspective. Copyright © 2017 Elsevier Inc. All rights reserved.

Correlative scanning-transmission electron microscopy reveals that a chimeric flavivirus is released as individual particles in secretory vesicles.

Directory of Open Access Journals (Sweden)

Julien Burlaud-Gaillard

Full Text Available The intracellular morphogenesis of flaviviruses has been well described, but flavivirus release from the host cell remains poorly documented. We took advantage of the optimized production of an attenuated chimeric yellow fever/dengue virus for vaccine purposes to study this phenomenon by microscopic approaches. Scanning electron microscopy (SEM showed the release of numerous viral particles at the cell surface through a short-lived process. For transmission electron microscopy (TEM studies of the intracellular ultrastructure of the small number of cells releasing viral particles at a given time, we developed a new correlative microscopy method: CSEMTEM (for correlative scanning electron microscopy - transmission electron microscopy. CSEMTEM analysis suggested that chimeric flavivirus particles were released as individual particles, in small exocytosis vesicles, via a regulated secretory pathway. Our morphological findings provide new insight into interactions between flaviviruses and cells and demonstrate that CSEMTEM is a useful new method, complementary to SEM observations of biological events by intracellular TEM investigations.

Recent achievements in multi-keV x-ray microscopy

International Nuclear Information System (INIS)

Susini, J.; Barrett, R.; Salome, M.; Kaulich, B.

2002-01-01

X-ray microscopy (XRM) techniques are emerging as powerful and complementary tools for sub-micron investigations. Soft XRM traditionally offers the possibility to form direct images of thick hydrated biological material in near-native environments, at a spatial resolution well beyond that achievable with visible light microscopy. Natural contrast is available in the soft X-ray region, in the so-called 'water-window', due to the presence of absorption edges of the major constituents (C,N,O). Recent advances in manufacturing techniques have enlarged the accessible energy range of micro-focusing optics and offer new applications in a broad range of disciplines. XRM in the 1-20 keV energy range is better suited to map trace elements in fluorescence yield, 3-D tomographic imaging and in micro-diffraction. After a brief introduction to the principles and methods, the main attributes of X-ray microscopy will be presented. This presentation will be biased towards sub-micron microscopy developed at the ESRF in the 2-10 keV energy. Strengths and weaknesses of X-ray microscopy and spectro-microscopy techniques will be discussed and illustrated by examples in biology, materials sciences and geology. (authors)

NDE Acoustic Microscopy Research Laboratory

Data.gov (United States)

Federal Laboratory Consortium — The purpose is to develop advanced, more effective high-resolution micro-NDE materials characterization methods using scanning acoustic microscopy. The laboratory's...

Fluorescent dyes with large Stokes shifts for super-resolution optical microscopy of biological objects: a review

International Nuclear Information System (INIS)

Sednev, Maksim V; Belov, Vladimir N; Hell, Stefan W

2015-01-01

The review deals with commercially available organic dyes possessing large Stokes shifts and their applications as fluorescent labels in optical microscopy based on stimulated emission depletion (STED). STED microscopy breaks Abbe’s diffraction barrier and provides optical resolution beyond the diffraction limit. STED microscopy is non-invasive and requires photostable fluorescent markers attached to biomolecules or other objects of interest. Up to now, in most biology-related STED experiments, bright and photoresistant dyes with small Stokes shifts of 20–40 nm were used. The rapid progress in STED microscopy showed that organic fluorophores possessing large Stokes shifts are indispensable in multi-color super-resolution techniques. The ultimate result of the imaging relies on the optimal combination of a dye, the bio-conjugation procedure and the performance of the optical microscope. Modern bioconjugation methods, basics of STED microscopy, as well as structures and spectral properties of the presently available fluorescent markers are reviewed and discussed. In particular, the spectral properties of the commercial dyes are tabulated and correlated with the available depletion wavelengths found in STED microscopes produced by LEICA Microsytems, Abberior Instruments and Picoquant GmbH. (topical review)

Atom probe field ion microscopy and related topics: A bibliography 1992

Energy Technology Data Exchange (ETDEWEB)

Russell, K.F.; Godfrey, R.D.; Miller, M.K.

1993-12-01

This bibliography contains citations of books, conference proceedings, journals, and patents published in 1992 on the following types of microscopy: atom probe field ion microscopy (108 items); field emission microscopy (101 items); and field ion microscopy (48 items). An addendum of 34 items missed in previous bibliographies is included.

Ballistic hole magnetic microscopy

NARCIS (Netherlands)

Haq, E.; Banerjee, T.; Siekman, M.H.; Lodder, J.C.; Jansen, R.

2005-01-01

A technique to study nanoscale spin transport of holes is presented: ballistic hole magnetic microscopy. The tip of a scanning tunneling microscope is used to inject hot electrons into a ferromagnetic heterostructure, where inelastic decay creates a distribution of electron-hole pairs.

Addressing preservation of elastic contrast in energy-filtered transmission electron microscopy

Energy Technology Data Exchange (ETDEWEB)

Brown, H.G.; D' Alfonso, A.J.; Forbes, B.D.; Allen, L.J., E-mail: lja@unimelb.edu.au

2016-01-15

Energy-filtered transmission electron microscopy (EFTEM) images with resolutions of the order of an Ångström can be obtained using modern microscopes corrected for chromatic aberration. However, the delocalized nature of the transition potentials for atomic ionization often confounds direct interpretation of EFTEM images, leading to what is known as “preservation of elastic contrast”. In this paper we demonstrate how more interpretable images might be obtained by scanning with a focused coherent probe and incoherently averaging the energy-filtered images over probe position. We dub this new imaging technique energy-filtered imaging scanning transmission electron microscopy (EFISTEM). We develop a theoretical framework for EFISTEM and show that it is in fact equivalent to precession EFTEM, where the plane wave illumination is precessed through a range of tilts spanning the same range of angles as the probe forming aperture in EFISTEM. It is demonstrated that EFISTEM delivers similar results to scanning transmission electron microscopy with an electron energy-loss spectrometer but has the advantage that it is immune to coherent aberrations and spatial incoherence of the probe and is also more resilient to scan distortions. - Highlights: • Interpretation of EFTEM images is complicated by preservation of elastic contrast. • More direct images obtained by scanning with a focused coherent probe and averaging. • This is equivalent to precession EFTEM through the solid angle defined by the probe. • Also yields similar results to energy-loss scanning transmission electron microscopy. • Scanning approach immune to probe aberrations and resilient to scan distortions.

Agrin is a major heparan sulfate proteoglycan in the human glomerular basement membrane.

Science.gov (United States)

Groffen, A J; Ruegg, M A; Dijkman, H; van de Velden, T J; Buskens, C A; van den Born, J; Assmann, K J; Monnens, L A; Veerkamp, J H; van den Heuvel, L P

1998-01-01

Agrin is a heparan sulfate proteoglycan (HSPG) that is highly concentrated in the synaptic basal lamina at the neuromuscular junction (NMJ). Agrin-like immunoreactivity is also detected outside the NMJ. Here we show that agrin is a major HSPG component of the human glomerular basement membrane (GBM). This is in addition to perlecan, a previously characterized HSPG of basement membranes. Antibodies against agrin and against an unidentified GBM HSPG produced a strong staining of the GBM and the NMJ, different from that observed with anti-perlecan antibodies. In addition, anti-agrin antisera recognized purified GBM HSPG and competed with an anti-GBM HSPG monoclonal antibody in ELISA. Furthermore, both antibodies recognized a molecule that migrated in SDS-PAGE as a smear and had a molecular mass of approximately 200-210 kD after deglycosylation. In immunoelectron microscopy, agrin showed a linear distribution along the GBM and was present throughout the width of the GBM. This was again different from perlecan, which was exclusively present on the endothelial side of the GBM and was distributed in a nonlinear manner. Quantitative ELISA showed that, compared with perlecan, the agrin-like GBM HSPG showed a sixfold higher molarity in crude glomerular extract. These results show that agrin is a major component of the GBM, indicating that it may play a role in renal ultrafiltration and cell matrix interaction. (J Histochem Cytochem 46:19-27, 1998)

Biostatistical analysis of quantitative immunofluorescence microscopy images.

Science.gov (United States)

Giles, C; Albrecht, M A; Lam, V; Takechi, R; Mamo, J C

2016-12-01

Semiquantitative immunofluorescence microscopy has become a key methodology in biomedical research. Typical statistical workflows are considered in the context of avoiding pseudo-replication and marginalising experimental error. However, immunofluorescence microscopy naturally generates hierarchically structured data that can be leveraged to improve statistical power and enrich biological interpretation. Herein, we describe a robust distribution fitting procedure and compare several statistical tests, outlining their potential advantages/disadvantages in the context of biological interpretation. Further, we describe tractable procedures for power analysis that incorporates the underlying distribution, sample size and number of images captured per sample. The procedures outlined have significant potential for increasing understanding of biological processes and decreasing both ethical and financial burden through experimental optimization. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

Networking strategies of the microscopy community for improved utilization of advanced instruments: (1) The Australian Microscopy and Microanalysis Research Facility (AMMRF)

International Nuclear Information System (INIS)

Ringer, S.P.; Apperley, M.H.

2014-01-01

This paper describes the strategy underpinning the formation and operation of the Australian Microscopy and Microanalysis Research Facility (AMMRF). AMMRF is a formal collaboration that links eight Australian Universities together to create a user-focused national capability in microscopy and microanalysis. The AMMRF flagship capabilities include: Cameca IMS-1280 and NanoSIMS-50 ion microprobes (University of Western Australia); High-throughput, high-resolution cryoTEM (University of Queensland); Atom Probe Microscopy (University of Sydney); High-resolution Focussed Ion-Beam and SEM (Universities of Adelaide and NSW); High-resolution SEM microanalysis facility (University of New South Wales); and PHI TRIFT V nanoToF ToF-SIMS (University of South Australia). Secondly, a network of peer support and expert training has been established amongst facility professional support staff. The governance and funding of the organisation are described and the advantages and achievements of a nationally coordinated facility for microscopy and microanalysis are set out. Selected data are presented that benchmark the performance of the facility, describe the economic impact and demonstrate the impact on the quality of research outcomes as a result of operating national collaborative research infrastructure for microscopy and microanalysis

Magnetic Resonance Force Microscopy System

Data.gov (United States)

Federal Laboratory Consortium — The Magnetic Resonance Force Microscopy (MRFM) system, developed by ARL, is the world's most sensitive nuclear magnetic resonance (NMR) spectroscopic analysis tool,...

Radiation defects in Te-implanted germanium. Electron microscopy and computer simulation studies

International Nuclear Information System (INIS)

Kalitzova, M.G.; Karpuzov, D.S.; Pashov, N.K.

1985-01-01

Direct observation of radiation damage induced by heavy ion implantation in crystalline germanium by means of high-resolution electron microscopy is reported. The dark-field lattice imaging mode is used, under conditions suitable for object-like imaging. Conventional TEM is used for estimating the efficiency of creating visibly damaged regions. Heavy ion damage clusters with three types of inner structure are observed: with near-perfect crystalline cores, and with metastable and stable amorphous cores. The MARLOWE computer code is used to simulate the atomic collision cascades and to obtain the lateral spread distributions of point defects created. A comparison of high-resolution electron microscopy (HREM) with computer simulation results shows encouraging agreement for the average cluster dimensions and for the lateral spread of vacancies and interstitials. (author)

QUANTITATIVE CONFOCAL LASER SCANNING MICROSCOPY

Directory of Open Access Journals (Sweden)

Merete Krog Raarup

2011-05-01

Full Text Available This paper discusses recent advances in confocal laser scanning microscopy (CLSM for imaging of 3D structure as well as quantitative characterization of biomolecular interactions and diffusion behaviour by means of one- and two-photon excitation. The use of CLSM for improved stereological length estimation in thick (up to 0.5 mm tissue is proposed. The techniques of FRET (Fluorescence Resonance Energy Transfer, FLIM (Fluorescence Lifetime Imaging Microscopy, FCS (Fluorescence Correlation Spectroscopy and FRAP (Fluorescence Recovery After Photobleaching are introduced and their applicability for quantitative imaging of biomolecular (co-localization and trafficking in live cells described. The advantage of two-photon versus one-photon excitation in relation to these techniques is discussed.

Channeling in helium ion microscopy: Mapping of crystal orientation

Directory of Open Access Journals (Sweden)

Vasilisa Veligura

2012-07-01

Full Text Available Background: The unique surface sensitivity and the high resolution that can be achieved with helium ion microscopy make it a competitive technique for modern materials characterization. As in other techniques that make use of a charged particle beam, channeling through the crystal structure of the bulk of the material can occur.Results: Here, we demonstrate how this bulk phenomenon affects secondary electron images that predominantly contain surface information. In addition, we will show how it can be used to obtain crystallographic information. We will discuss the origin of channeling contrast in secondary electron images, illustrate this with experiments, and develop a simple geometric model to predict channeling maxima.Conclusion: Channeling plays an important role in helium ion microscopy and has to be taken into account when trying to achieve maximum image quality in backscattered helium images as well as secondary electron images. Secondary electron images can be used to extract crystallographic information from bulk samples as well as from thin surface layers, in a straightforward manner.

Enhanced light microscopy visualization of virus particles from Zika virus to filamentous ebolaviruses.

Directory of Open Access Journals (Sweden)

George G Daaboul

Full Text Available Light microscopy is a powerful tool in the detection and analysis of parasites, fungi, and prokaryotes, but has been challenging to use for the detection of individual virus particles. Unlabeled virus particles are too small to be visualized using standard visible light microscopy. Characterization of virus particles is typically performed using higher resolution approaches such as electron microscopy or atomic force microscopy. These approaches require purification of virions away from their normal millieu, requiring significant levels of expertise, and can only enumerate small numbers of particles per field of view. Here, we utilize a visible light imaging approach called Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS that allows automated counting and sizing of thousands of individual virions. Virions are captured directly from complex solutions onto a silicon chip and then detected using a reflectance interference imaging modality. We show that the use of different imaging wavelengths allows the visualization of a multitude of virus particles. Using Violet/UV illumination, the SP-IRIS technique is able to detect individual flavivirus particles (~40 nm, while green light illumination is capable of identifying and discriminating between vesicular stomatitis virus and vaccinia virus (~360 nm. Strikingly, the technology allows the clear identification of filamentous infectious ebolavirus particles and virus-like particles. The ability to differentiate and quantify unlabeled virus particles extends the usefulness of traditional light microscopy and can be embodied in a straightforward benchtop approach allowing widespread applications ranging from rapid detection in biological fluids to analysis of virus-like particles for vaccine development and production.

Electron microscopy study of advanced heterostructures for optoelectronics

NARCIS (Netherlands)

Katcki, J.; Ratajczak, J.; Phillipp, F.; Muszalski, J.; Bugajski, M.; Chen, J.X.; Fiore, A.

2003-01-01

The application of cross-sectional transmission electron microscopy and SEM to the investigation of optoelectronic devices are reviewed. Special attention was paid to the electron microscopy assessment of the growth perfection of such crucial elements of the devices like quantum wells, quantum dots,

Optimisation-based wavefront sensorless adaptive optics for microscopy

NARCIS (Netherlands)

Antonello, J.

2014-01-01

Microscopy is an essential tool for life sciences. Thanks to the development of confocal and multiphoton microscopy, scientists are able to obtain high-resolution 3D views of biological specimens. Nevertheless, spatial variations in the index of refraction within specimens cause aberrations that

2nd International Multidisciplinary Microscopy and Microanalysis Congress

CERN Document Server

Oral, Ahmet; Ozer, Mehmet

2015-01-01

The 2nd International Multidisciplinary Microscopy and Microanalysis Congress & Exhibition (InterM 2014) was held on 16–19 October 2014 in Oludeniz, Fethiye/ Mugla, Turkey. The aim of the congress was to gather scientists from various branches and discuss the latest improvements in the field of microscopy. The focus of the congress has been widened in an "interdisciplinary" manner, so as to allow all scientists working on several related subjects to participate and present their work. These proceedings include 33 peer-reviewed technical papers, submitted by leading academic and research institutions from over 17 countries and representing some of the most cutting-edge research available. The papers were presented at the congress in the following sessions: ·         Applications of Microscopy in the Physical Sciences ·         Applications of Microscopy in the Biological Sciences.

Correlative Light-Electron Microscopy Shows RGD-Targeted ZnO Nanoparticles Dissolve in the Intracellular Environment of Triple Negative Breast Cancer Cells and Cause Apoptosis with Intratumor Heterogeneity

KAUST Repository

Othman, Basmah A.; Greenwood, Christina; AbuElela, Ayman; Bharath, Anil A.; Chen, Shu; Theodorou, Ioannis; Douglas, Trevor; Uchida, Maskai; Ryan, Mary; Merzaban, Jasmeen; Porter, Alexandra E.

2016-01-01

ZnO nanoparticles (NPs) are reported to show a high degree of cancer cell selectivity with potential use in cancer imaging and therapy. Questions remain about the mode by which the ZnO NPs cause cell death, whether they exert an intra- or extracellular effect, and the resistance among different cancer cell types to ZnO NP exposure. The present study quantifies the variability between the cellular toxicity, dynamics of cellular uptake, and dissolution of bare and RGD (Arg-Gly-Asp)-targeted ZnO NPs by MDA-MB-231 cells. Compared to bare ZnO NPs, RGD-targeting of the ZnO NPs to integrin αvβ3 receptors expressed on MDA-MB-231 cells appears to increase the toxicity of the ZnO NPs to breast cancer cells at lower doses. Confocal microscopy of live MDA-MB-231 cells confirms uptake of both classes of ZnO NPs with a commensurate rise in intracellular Zn2+ concentration prior to cell death. The response of the cells within the population to intracellular Zn2+ is highly heterogeneous. In addition, the results emphasize the utility of dynamic and quantitative imaging in understanding cell uptake and processing of targeted therapeutic ZnO NPs at the cellular level by heterogeneous cancer cell populations, which can be crucial for the development of optimized treatment strategies.

Correlative Light-Electron Microscopy Shows RGD-Targeted ZnO Nanoparticles Dissolve in the Intracellular Environment of Triple Negative Breast Cancer Cells and Cause Apoptosis with Intratumor Heterogeneity

KAUST Repository

Othman, Basmah A.

2016-04-01

ZnO nanoparticles (NPs) are reported to show a high degree of cancer cell selectivity with potential use in cancer imaging and therapy. Questions remain about the mode by which the ZnO NPs cause cell death, whether they exert an intra- or extracellular effect, and the resistance among different cancer cell types to ZnO NP exposure. The present study quantifies the variability between the cellular toxicity, dynamics of cellular uptake, and dissolution of bare and RGD (Arg-Gly-Asp)-targeted ZnO NPs by MDA-MB-231 cells. Compared to bare ZnO NPs, RGD-targeting of the ZnO NPs to integrin αvβ3 receptors expressed on MDA-MB-231 cells appears to increase the toxicity of the ZnO NPs to breast cancer cells at lower doses. Confocal microscopy of live MDA-MB-231 cells confirms uptake of both classes of ZnO NPs with a commensurate rise in intracellular Zn2+ concentration prior to cell death. The response of the cells within the population to intracellular Zn2+ is highly heterogeneous. In addition, the results emphasize the utility of dynamic and quantitative imaging in understanding cell uptake and processing of targeted therapeutic ZnO NPs at the cellular level by heterogeneous cancer cell populations, which can be crucial for the development of optimized treatment strategies.

EVALUATION OF THE ULTRASTRUCTURE OF THE SMALL INTESTINE OF HIV INFECTED CHILDREN BY TRANSMISSION AND SCANNING ELECTRONIC MICROSCOPY

Directory of Open Access Journals (Sweden)

Christiane Araujo Chaves LEITE

2013-03-01

Full Text Available Objectives To describe HIV children's small intestinal ultrastructural findings. Methods Descriptive, observational study of small intestine biopsies performed between August 1994 and May 1995 at São Paulo, SP, Brazil. This material pertained to 11 HIV infected children and was stored in a laboratory in paraffin blocks. Scanning and transmission electronic microscopy were used to view those intestine samples and ultrastructural findings were described by analyzing digitalized photos of this material. Ethical Committee approval was obtained. Results In most samples scanning microscopy showed various degrees of shortening and decreasing number of microvilli and also completes effacements in some areas. Derangement of the enterocytes was seen frequently and sometimes cells well defined borders limits seemed to be loosened. In some areas a mucous-fibrin like membrane with variable thickness and extension appeared to partially or totally coat the epithelial surface. Fat drops were present in the intestinal lumen in various samples and a bacterium morphologically resembling bacilli was seen in two occasions. Scanning microscopy confirmed transmission microscopy microvilli findings and also showed little “tufts” of those structures. In addition, it showed an increased number of vacuoles and multivesicular bodies inside various enterocytes, an increased presence of intraepithelial lymphocytes, mitochondrial vacuolization and basement membrane enlargement in the majority of samples analyzed. However, some samples exhibited normal aspect. Conclusions Our study showed the common occurrence of various important intestinal ultrastructural alterations with variable degrees among HIV infected children, some of them in our knowledge not described before.

Quadratic electromechanical strain in silicon investigated by scanning probe microscopy

Science.gov (United States)

Yu, Junxi; Esfahani, Ehsan Nasr; Zhu, Qingfeng; Shan, Dongliang; Jia, Tingting; Xie, Shuhong; Li, Jiangyu

2018-04-01

Piezoresponse force microscopy (PFM) is a powerful tool widely used to characterize piezoelectricity and ferroelectricity at the nanoscale. However, it is necessary to distinguish microscopic mechanisms between piezoelectricity and non-piezoelectric contributions measured by PFM. In this work, we systematically investigate the first and second harmonic apparent piezoresponses of a silicon wafer in both vertical and lateral modes, and we show that it exhibits an apparent electromechanical response that is quadratic to the applied electric field, possibly arising from ionic electrochemical dipoles induced by the charged probe. As a result, the electromechanical response measured is dominated by the second harmonic response in the vertical mode, and its polarity can be switched by the DC voltage with the evolving coercive field and maximum amplitude, in sharp contrast to typical ferroelectric materials we used as control. The ionic activity in silicon is also confirmed by the scanning thermo-ionic microscopy measurement, and the work points toward a set of methods to distinguish true piezoelectricity from the apparent ones.

Soft X-ray microscopy and lithography with synchrotron radiation

International Nuclear Information System (INIS)

Gudat, W.

1977-12-01

Considerable progress in the technique microscopy with soft X-ray radiation has been achieved in particular through the application of synchrotron radiation. Various methods which are currently being studied theoretically or already being used practically will be described briefly. Attention is focussed on the method of contact microscopy. Various biological specimens have been investigated by this method with a resolution as good as 100 A. X-ray lithography which in the technical procedure is very similar to contact microscopy gives promise for the fabrication of high quality submicron structures in electronic device production. Important factors limiting the resolution and determining the performance of contact microscopy and X-ray lithography will be discussed. (orig.) [de

Scanning transmission electron microscopy imaging and analysis

CERN Document Server

Pennycook, Stephen J

2011-01-01

Provides the first comprehensive treatment of the physics and applications of this mainstream technique for imaging and analysis at the atomic level Presents applications of STEM in condensed matter physics, materials science, catalysis, and nanoscience Suitable for graduate students learning microscopy, researchers wishing to utilize STEM, as well as for specialists in other areas of microscopy Edited and written by leading researchers and practitioners

Scanning photoemission microscopy with synchrotron radiation

Science.gov (United States)

Ade, Harald W.

1992-08-01

Progress in photoemission spectro-microscopy at various synchrotron radiation facilities is reviewed. Microprobe devices such as MAXIMUM at the SRC in Wisconsin, the X1-SPEM at the NSLS at BNL, as well as the ellipsoidal ring mirror microscope at DESY in Hamburg, recorded first images during the last few years. The present status of these devices which achieve their lateral resolution by focusing X-rays to a small spot is the primary focus of this paper, but work representing other approaches to spectro-microscopy is also discussed.

Single-wavelength functional photoacoustic microscopy in biological tissue.

Science.gov (United States)

Danielli, Amos; Favazza, Christopher P; Maslov, Konstantin; Wang, Lihong V

2011-03-01

Recently, we developed a reflection-mode relaxation photoacoustic microscope, based on saturation intensity, to measure picosecond relaxation times using a nanosecond laser. Here, using the different relaxation times of oxygenated and deoxygenated hemoglobin molecules, both possessing extremely low fluorescence quantum yields, the oxygen saturation was quantified in vivo with single-wavelength photoacoustic microscopy. All previous functional photoacoustic microscopy measurements required imaging with multiple-laser-wavelength measurements to quantify oxygen saturation. Eliminating the need for multiwavelength measurements removes the influence of spectral properties on oxygenation calculations and improves the portability and cost-effectiveness of functional or molecular photoacoustic microscopy.

Intermittent contact atomic force microscopy in electrochemical environment

Energy Technology Data Exchange (ETDEWEB)

Haering, P; Koetz, R [Paul Scherrer Inst. (PSI), Villigen (Switzerland); Siegenthaler, H [Bern Univ., Bern (Switzerland)

1997-06-01

In situ measurements with Atomic Force Microscopy may cause surface modifications due to the tip-surface interactions. As an alternative and less destructive method, Intermittent Contact Atomic Force Microscopy (ICAFM) has been tested in an electrolytic environment. In the ICAFM mode the tip is not constantly in contact with the surface under investigation but is tapping onto the surface with a certain frequency. A commercial Park Scientific Instruments Microscopy has been modified to enable in situ experiment with ICAFM. It was possible to image iridium oxide films with ICAFM in the electrolytic environment without any noticeable surface modifications. (author) 3 figs., 4 refs.

Machine learning approach for single molecule localisation microscopy.

Science.gov (United States)

Colabrese, Silvia; Castello, Marco; Vicidomini, Giuseppe; Del Bue, Alessio

2018-04-01

Single molecule localisation (SML) microscopy is a fundamental tool for biological discoveries; it provides sub-diffraction spatial resolution images by detecting and localizing "all" the fluorescent molecules labeling the structure of interest. For this reason, the effective resolution of SML microscopy strictly depends on the algorithm used to detect and localize the single molecules from the series of microscopy frames. To adapt to the different imaging conditions that can occur in a SML experiment, all current localisation algorithms request, from the microscopy users, the choice of different parameters. This choice is not always easy and their wrong selection can lead to poor performance. Here we overcome this weakness with the use of machine learning. We propose a parameter-free pipeline for SML learning based on support vector machine (SVM). This strategy requires a short supervised training that consists in selecting by the user few fluorescent molecules (∼ 10-20) from the frames under analysis. The algorithm has been extensively tested on both synthetic and real acquisitions. Results are qualitatively and quantitatively consistent with the state of the art in SML microscopy and demonstrate that the introduction of machine learning can lead to a new class of algorithms competitive and conceived from the user point of view.

Metal layer mask patterning by force microscopy lithography

International Nuclear Information System (INIS)

Filho, H.D. Fonseca; Mauricio, M.H.P.; Ponciano, C.R.; Prioli, R.

2004-01-01

The nano-lithography of a metallic surface in air by atomic force microscopy while operated in contact mode and equipped with a diamond tip is presented. The aluminum mask was prepared by thermal deposition on arsenic sulfide films. The analysis of the scratches performed by the tip on the metallic mask show that the depth of the lithographed pattern increases with the increase of the applied normal force. The scanning velocity is also shown to influence the AFM patterning process. As the scanning velocity increases, the scratch depth and width decreases. Nano-indentations performed with the diamond tip show that the plastically deformed surface increases with the increase of the duration of the applied force. The use of the nano-lithography method to create nano-structures is discussed

Biological applications of phase-contrast electron microscopy.

Science.gov (United States)

Nagayama, Kuniaki

2014-01-01

Here, I review the principles and applications of phase-contrast electron microscopy using phase plates. First, I develop the principle of phase contrast based on a minimal model of microscopy, introducing a double Fourier-transform process to mathematically formulate the image formation. Next, I explain four phase-contrast (PC) schemes, defocus PC, Zernike PC, Hilbert differential contrast, and schlieren optics, as image-filtering processes in the context of the minimal model, with particular emphases on the Zernike PC and corresponding Zernike phase plates. Finally, I review applications of Zernike PC cryo-electron microscopy to biological systems such as protein molecules, virus particles, and cells, including single-particle analysis to delineate three-dimensional (3D) structures of protein and virus particles and cryo-electron tomography to reconstruct 3D images of complex protein systems and cells.

A novel multiphoton microscopy images segmentation method based on superpixel and watershed.

Science.gov (United States)

Wu, Weilin; Lin, Jinyong; Wang, Shu; Li, Yan; Liu, Mingyu; Liu, Gaoqiang; Cai, Jianyong; Chen, Guannan; Chen, Rong

2017-04-01

Multiphoton microscopy (MPM) imaging technique based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) shows fantastic performance for biological imaging. The automatic segmentation of cellular architectural properties for biomedical diagnosis based on MPM images is still a challenging issue. A novel multiphoton microscopy images segmentation method based on superpixels and watershed (MSW) is presented here to provide good segmentation results for MPM images. The proposed method uses SLIC superpixels instead of pixels to analyze MPM images for the first time. The superpixels segmentation based on a new distance metric combined with spatial, CIE Lab color space and phase congruency features, divides the images into patches which keep the details of the cell boundaries. Then the superpixels are used to reconstruct new images by defining an average value of superpixels as image pixels intensity level. Finally, the marker-controlled watershed is utilized to segment the cell boundaries from the reconstructed images. Experimental results show that cellular boundaries can be extracted from MPM images by MSW with higher accuracy and robustness. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Tip radius preservation for high resolution imaging in amplitude modulation atomic force microscopy

Energy Technology Data Exchange (ETDEWEB)

Ramos, Jorge R., E-mail: jorge.rr@cea.cu [Instituto de Ciencia de Materiales de Madrid, Sor Juana Inés de la Cruz 3, Canto Blanco, 28049 Madrid, España (Spain)

2014-07-28

The acquisition of high resolution images in atomic force microscopy (AFM) is correlated to the cantilever's tip shape, size, and imaging conditions. In this work, relative tip wear is quantified based on the evolution of a direct experimental observable in amplitude modulation atomic force microscopy, i.e., the critical amplitude. We further show that the scanning parameters required to guarantee a maximum compressive stress that is lower than the yield/fracture stress of the tip can be estimated via experimental observables. In both counts, the optimized parameters to acquire AFM images while preserving the tip are discussed. The results are validated experimentally by employing IgG antibodies as a model system.

Proceedings of 10. Conference on Electron Microscopy of Solids

International Nuclear Information System (INIS)

1999-01-01

The new technical solutions and methodical variants of electron microscopy i. e. transmission electron microscopy and scanning electron microscopy have been presented. Development of new methods and microscope constructions which became more and more sophisticated causes the progress in possible applications. The broad spectrum of such applications in metallurgy, materials science, chemical engineering, electronics, physical chemistry, solid state physics, mineralogy and other branches of science and technique have been performed and discussed at the conference

Reproducibility in light microscopy: Maintenance, standards and SOPs.

Science.gov (United States)

Deagle, Rebecca C; Wee, Tse-Luen Erika; Brown, Claire M

2017-08-01

Light microscopy has grown to be a valuable asset in both the physical and life sciences. It is a highly quantitative method available in individual research laboratories and often centralized in core facilities. However, although quantitative microscopy is becoming a customary tool in research, it is rarely standardized. To achieve accurate quantitative microscopy data and reproducible results, three levels of standardization must be considered: (1) aspects of the microscope, (2) the sample, and (3) the detector. The accuracy of the data is only as reliable as the imaging system itself, thereby imposing the need for routine standard performance testing. Depending on the task some maintenance procedures should be performed once a month, some before each imaging session, while others conducted annually. This text should be implemented as a resource for researchers to integrate with their own standard operating procedures to ensure the highest quality quantitative microscopy data. Copyright © 2017. Published by Elsevier Ltd.

Imaging a seizure model in zebrafish with structured illumination light sheet microscopy

Science.gov (United States)

Liu, Yang; Dale, Savannah; Ball, Rebecca; VanLeuven, Ariel J.; Baraban, Scott; Sornborger, Andrew; Lauderdale, James D.; Kner, Peter

2018-02-01

Zebrafish are a promising vertebrate model for elucidating how neural circuits generate behavior under normal and pathological conditions. The Baraban group first demonstrated that zebrafish larvae are valuable for investigating seizure events and can be used as a model for epilepsy in humans. Because of their small size and transparency, zebrafish embryos are ideal for imaging seizure activity using calcium indicators. Light-sheet microscopy is well suited to capturing neural activity in zebrafish because it is capable of optical sectioning, high frame rates, and low excitation intensities. We describe work in our lab to use light-sheet microscopy for high-speed long-time imaging of neural activity in wildtype and mutant zebrafish to better understand the connectivity and activity of inhibitory neural networks when GABAergic signaling is altered in vivo. We show that, with light-sheet microscopy, neural activity can be recorded at 23 frames per second in twocolors for over 10 minutes allowing us to capture rare seizure events in mutants. We have further implemented structured illumination to increase resolution and contrast in the vertical and axial directions during high-speed imaging at an effective frame rate of over 7 frames per second.

A novel method for enhancing the lateral resolution and image SNR in confocal microscopy

Science.gov (United States)

Chen, Youhua; Zhu, Dazhao; Fang, Yue; Kuang, Cuifang; Liu, Xu

2017-12-01

There is always a tradeoff between the resolution and the signal-to-noise ratio (SNR) in confocal microscopy. In particular, the pinhole size is very important for maintaining a balance between them. In this paper, we propose a method for improving the lateral resolution and image SNR in confocal microscopy without making any changes to the hardware. By using the fluorescence emission difference (FED) approach, we divide the images acquired by different pinhole sizes into one image acquired by the central pinhole and several images acquired by ring-shaped pinholes. Then, they are added together with the deconvolution method. Simulation and experimental results for fluorescent particles and cells show that our method can achieve a far better resolution than a large pinhole and a higher SNR than a small pinhole. Moreover, our method can improve the performance of classic confocal laser scanning microscopy (CLSM) to a certain extent, especially CLSM with a continuously variable pinhole.

Virtual Hematoxylin and Eosin Transillumination Microscopy Using Epi-Fluorescence Imaging.

Science.gov (United States)

Giacomelli, Michael G; Husvogt, Lennart; Vardeh, Hilde; Faulkner-Jones, Beverly E; Hornegger, Joachim; Connolly, James L; Fujimoto, James G

2016-01-01

We derive a physically realistic model for the generation of virtual transillumination, white light microscopy images using epi-fluorescence measurements from thick, unsectioned tissue. We demonstrate this technique by generating virtual transillumination H&E images of unsectioned human breast tissue from epi-fluorescence multiphoton microscopy data. The virtual transillumination algorithm is shown to enable improved contrast and color accuracy compared with previous color mapping methods. Finally, we present an open source implementation of the algorithm in OpenGL, enabling real-time GPU-based generation of virtual transillumination microscopy images using conventional fluorescence microscopy systems.

Brillouin microscopy: assessing ocular tissue biomechanics.

Science.gov (United States)

Yun, Seok Hyun; Chernyak, Dimitri

2018-07-01

Assessment of corneal biomechanics has been an unmet clinical need in ophthalmology for many years. Many researchers and clinicians have identified corneal biomechanics as source of variability in refractive procedures and one of the main factors in keratoconus. However, it has been difficult to accurately characterize corneal biomechanics in patients. The recent development of Brillouin light scattering microscopy heightens the promise of bringing biomechanics into the clinic. The aim of this review is to overview the progress and discuss prospective applications of this new technology. Brillouin microscopy uses a low-power near-infrared laser beam to determine longitudinal modulus or mechanical compressibility of tissue by analyzing the return signal spectrum. Human clinical studies have demonstrated significant difference in the elastic properties of normal corneas versus corneas diagnosed with mild and severe keratoconus. Clinical data have also shown biomechanical changes after corneal cross-linking treatment of keratoconus patients. Brillouin measurements of the crystalline lens and sclera have also been demonstrated. Brillouin microscopy is a promising technology under commercial development at present. The technique enables physicians to characterize the biomechanical properties of ocular tissues.

Label-free imaging of gold nanoparticles in single live cells by photoacoustic microscopy

Science.gov (United States)

Tian, Chao; Qian, Wei; Shao, Xia; Xie, Zhixing; Cheng, Xu; Liu, Shengchun; Cheng, Qian; Liu, Bing; Wang, Xueding

2016-03-01

Gold nanoparticles (AuNPs) have been extensively explored as a model nanostructure in nanomedicine and have been widely used to provide advanced biomedical research tools in diagnostic imaging and therapy. Due to the necessity of targeting AuNPs to individual cells, evaluation and visualization of AuNPs in the cellular level is critical to fully understand their interaction with cellular environment. Currently imaging technologies, such as fluorescence microscopy and transmission electron microscopy all have advantages and disadvantages. In this paper, we synthesized AuNPs by femtosecond pulsed laser ablation, modified their surface chemistry through sequential bioconjugation, and targeted the functionalized AuNPs with individual cancer cells. Based on their high optical absorption contrast, we developed a novel, label-free imaging method to evaluate and visualize intracellular AuNPs using photoacoustic microscopy (PAM). Preliminary study shows that the PAM imaging technique is capable of imaging cellular uptake of AuNPs in vivo at single-cell resolution, which provide an important tool for the study of AuNPs in nanomedicine.

Gastric Tissue Damage Analysis Generated by Ischemia: Bioimpedance, Confocal Endomicroscopy, and Light Microscopy

Directory of Open Access Journals (Sweden)

Nohra E. Beltran

2013-01-01

Full Text Available The gastric mucosa ischemic tissular damage plays an important role in critical care patients’ outcome, because it is the first damaged tissue by compensatory mechanism during shock. The aim of the study is to relate bioimpedance changes with tissular damage level generated by ischemia by means of confocal endomicroscopy and light microscopy. Bioimpedance of the gastric mucosa and confocal images were obtained from Wistar male rats during basal and ischemia conditions. They were anesthetized, and stain was applied (fluorescein and/or acriflavine. The impedance spectroscopy catheter was inserted and then confocal endomicroscopy probe. After basal measurements and biopsy, hepatic and gastric arteries clamping induced ischemia. Finally, pyloric antrum tissue was preserved in buffered formaldehyde (10% for histology processing using light microscopy. Confocal images were equalized, binarized, and boundary defined, and infiltrations were quantified. Impedance and infiltrations increased with ischemia showing significant changes between basal and ischemia conditions (. Light microscopy analysis allows detection of general alterations in cellular and tissular integrity, confirming gastric reactance and confocal images quantification increments obtained during ischemia.

Effectiveness of Modal Decomposition for Tapping Atomic Force Microscopy Microcantilevers in Liquid Environment.

Science.gov (United States)

Kim, Il Kwang; Lee, Soo Il

2016-05-01

The modal decomposition of tapping mode atomic force microscopy microcantilevers in liquid environments was studied experimentally. Microcantilevers with different lengths and stiffnesses and two sample surfaces with different elastic moduli were used in the experiment. The response modes of the microcantilevers were extracted as proper orthogonal modes through proper orthogonal decomposition. Smooth orthogonal decomposition was used to estimate the resonance frequency directly. The effects of the tapping setpoint and the elastic modulus of the sample under test were examined in terms of their multi-mode responses with proper orthogonal modes, proper orthogonal values, smooth orthogonal modes and smooth orthogonal values. Regardless of the stiffness of the microcantilever under test, the first mode was dominant in tapping mode atomic force microscopy under normal operating conditions. However, at lower tapping setpoints, the flexible microcantilever showed modal distortion and noise near the tip when tapping on a hard sample. The stiff microcantilever had a higher mode effect on a soft sample at lower tapping setpoints. Modal decomposition for tapping mode atomic force microscopy can thus be used to estimate the characteristics of samples in liquid environments.

Scanning tunneling microscopy and spectroscopy on GaN and InGaN surfaces

International Nuclear Information System (INIS)

Krueger, David

2009-01-01

Optelectronic devices based on gallium nitride (GaN) and indium gallium nitride (InGaN) are in the focus of research since more than 20 years and still have great potential for optical applications. In the first part of this work non-polar surfaces of GaN are investigated by scanning electron microscopy (SEM), atomic force microscopy (AFM) and scanning tunneling microscopy (STM). In SEM and AFM, the (1 anti 100)- and especially the (anti 2110)-plane are quite corrugated. For the first time, the (anti 2110)-plane of GaN is atomically resolved in STM. In the second part InGaN quantum dot layers are investigated by X-ray photoelectron spectroscopy (XPS), scanning tunneling spectroscopy (STS) and STM. The STMmeasurements show the dependency of surface morphology on growth conditions in the metalorganic vapour phase epitaxy (MOVPE). Nucleation, a new MOVPE-strategy, is based on phase separations on surfaces. It is shown that locally varying density of states and bandgaps can be detected by STS, that means bandgap histograms and 2D-bandgap-mapping. (orig.)

Wave front engineering by means of diffractive optical elements for applications in microscopy

Science.gov (United States)

Cojoc, Dan; Ferrari, Enrico; Garbin, Valeria; Cabrini, Stefano; Carpentiero, Alessandro; Prasciolu, Mauro; Businaro, Luca; Kaulich, Burchard; Di Fabrizio, Enzo

2006-05-01

We present a unified view regarding the use of diffractive optical elements (DOEs) for microscopy applications a wide range of electromagnetic spectrum. The unified treatment is realized through the design and fabrication of DOE through which wave front beam shaping is obtained. In particular we show applications ranging from micromanipulation using optical tweezers to X-ray differential interference contrast (DIC) microscopy. We report some details on the design and physical implementation of diffractive elements that beside focusing perform also other optical functions: beam splitting, beam intensity and phase redistribution or mode conversion. Laser beam splitting is used for multiple trapping and independent manipulation of spherical micro beads and for direct trapping and manipulation of biological cells with non-spherical shapes. Another application is the Gauss to Laguerre-Gaussian mode conversion, which allows to trap and transfer orbital angular momentum of light to micro particles with high refractive index and to trap and manipulate low index particles. These experiments are performed in an inverted optical microscope coupled with an infrared laser beam and a spatial light modulator for DOEs implementation. High resolution optics, fabricated by means of e-beam lithography, are demonstrated to control the intensity and the phase of the sheared beams in X-ray DIC microscopy. DIC experiments with phase objects reveal a dramatic increase in image contrast compared to bright-field X-ray microscopy.

Surface chemistry and microscopy of food powders

Science.gov (United States)

Burgain, Jennifer; Petit, Jeremy; Scher, Joël; Rasch, Ron; Bhandari, Bhesh; Gaiani, Claire

2017-12-01

Despite high industrial and scientific interest, a comprehensive review of the surface science of food powders is still lacking. There is a real gap between scientific concerns of the field and accessible reviews on the subject. The global description of the surface of food powders by multi-scale microscopy approaches seems to be essential in order to investigate their complexity and take advantage of their high innovation potential. Links between these techniques and the interest to develop a multi-analytical approach to investigate scientific questions dealing with powder functionality are discussed in the second part of the review. Finally, some techniques used in others fields and showing promising possibilities in the food powder domain will be highlighted.

Hierarchical super-structure identified by polarized light microscopy, electron microscopy and nanoindentation: Implications for the limits of biological control over the growth mode of abalone sea shells

Directory of Open Access Journals (Sweden)

Schneider Andreas S

2012-09-01

Full Text Available Abstract Background Mollusc shells are commonly investigated using high-resolution imaging techniques based on cryo-fixation. Less detailed information is available regarding the light-optical properties. Sea shells of Haliotis pulcherina were embedded for polishing in defined orientations in order to investigate the interface between prismatic calcite and nacreous aragonite by standard materialographic methods. A polished thin section of the interface was prepared with a defined thickness of 60 μm for quantitative birefringence analysis using polarized light and LC-PolScope microscopy. Scanning electron microscopy images were obtained for comparison. In order to study structural-mechanical relationships, nanoindentation experiments were performed. Results Incident light microscopy revealed a super-structure in semi-transparent regions of the polished cross-section under a defined angle. This super-structure is not visible in transmitted birefringence analysis due to the blurred polarization of small nacre platelets and numerous organic interfaces. The relative orientation and homogeneity of calcite prisms was directly identified, some of them with their optical axes exactly normal to the imaging plane. Co-oriented "prism colonies" were identified by polarized light analyses. The nacreous super-structure was also visualized by secondary electron imaging under defined angles. The domains of the super-structure were interpreted to consist of crystallographically aligned platelet stacks. Nanoindentation experiments showed that mechanical properties changed with the same periodicity as the domain size. Conclusions In this study, we have demonstrated that insights into the growth mechanisms of nacre can be obtained by conventional light-optical methods. For example, we observed super-structures formed by co-oriented nacre platelets as previously identified using X-ray Photo-electron Emission Microscopy (X-PEEM [Gilbert et al., Journal of the

Identification of nodal tissue in the living heart using rapid scanning fiber-optics confocal microscopy and extracellular fluorophores.

Science.gov (United States)

Huang, Chao; Kaza, Aditya K; Hitchcock, Robert W; Sachse, Frank B

2013-09-01

Risks associated with pediatric reconstructive heart surgery include injury of the sinoatrial node (SAN) and atrioventricular node (AVN), requiring cardiac rhythm management using implantable pacemakers. These injuries are the result of difficulties in identifying nodal tissues intraoperatively. Here we describe an approach based on confocal microscopy and extracellular fluorophores to quantify tissue microstructure and identify nodal tissue. Using conventional 3-dimensional confocal microscopy we investigated the microstructural arrangement of SAN, AVN, and atrial working myocardium (AWM) in fixed rat heart. AWM exhibited a regular striated arrangement of the extracellular space. In contrast, SAN and AVN had an irregular, reticulated arrangement. AWM, SAN, and AVN tissues were beneath a thin surface layer of tissue that did not obstruct confocal microscopic imaging. Subsequently, we imaged tissues in living rat hearts with real-time fiber-optics confocal microscopy. Fiber-optics confocal microscopy images resembled images acquired with conventional confocal microscopy. We investigated spatial regularity of tissue microstructure from Fourier analysis and second-order image moments. Fourier analysis of fiber-optics confocal microscopy images showed that the spatial regularity of AWM was greater than that of nodal tissues (37.5 ± 5.0% versus 24.3 ± 3.9% for SAN and 23.8 ± 3.7% for AVN; Pfiber-optics confocal microscopy. Application of the approach in pediatric reconstructive heart surgery may reduce risks of injuring nodal tissues.

Simultaneous correlative scanning electron and high-NA fluorescence microscopy.

Directory of Open Access Journals (Sweden)

Nalan Liv

Full Text Available Correlative light and electron microscopy (CLEM is a unique method for investigating biological structure-function relations. With CLEM protein distributions visualized in fluorescence can be mapped onto the cellular ultrastructure measured with electron microscopy. Widespread application of correlative microscopy is hampered by elaborate experimental procedures related foremost to retrieving regions of interest in both modalities and/or compromises in integrated approaches. We present a novel approach to correlative microscopy, in which a high numerical aperture epi-fluorescence microscope and a scanning electron microscope illuminate the same area of a sample at the same time. This removes the need for retrieval of regions of interest leading to a drastic reduction of inspection times and the possibility for quantitative investigations of large areas and datasets with correlative microscopy. We demonstrate Simultaneous CLEM (SCLEM analyzing cell-cell connections and membrane protrusions in whole uncoated colon adenocarcinoma cell line cells stained for actin and cortactin with AlexaFluor488. SCLEM imaging of coverglass-mounted tissue sections with both electron-dense and fluorescence staining is also shown.

Microscopy as a diagnostic tool in pulmonary tuberculosis

Directory of Open Access Journals (Sweden)

Ritu Singhal

2015-01-01

Full Text Available Tuberculosis continues to cast a huge impact on humanity with its high incidence and mortality, especially in developing countries. For tuberculosis case detection, microscopy continues to be indispensible, given its low cost, rapidity, simplicity of procedure and high specificity. Modifications have attempted to improve the sensitivity of microscopy which include: concentration methods such as centrifugation, N-acetyl cysteine–sodium hydroxide, bleach, ammonium sulfate or chitin. Furthermore, classical Ziehl–Neelsen (ZN staining has been subjected to varying carbol fuchsin concentrations or replaced by Kinyoun staining, fluorescent microscopy or immune-fluorescence. Currently, light emitting diode fluorescence is recognizably the most plausible method as an alternative to ZN staining.

Digital Fourier microscopy for soft matter dynamics

International Nuclear Information System (INIS)

Giavazzi, Fabio; Cerbino, Roberto

2014-01-01

Soft matter is studied with a large portfolio of methods. Light scattering and video microscopy are the most employed at optical wavelengths. Light scattering provides ensemble-averaged information on soft matter in the reciprocal space. The wave-vectors probed correspond to length scales ranging from a few nanometers to fractions of millimetre. Microscopy probes the sample directly in the real space, by offering a unique access to the local properties. However, optical resolution issues limit the access to length scales smaller than approximately 200 nm. We describe recent work that bridges the gap between scattering and microscopy. Several apparently unrelated techniques are found to share a simple basic idea: the correlation properties of the sample can be characterized in the reciprocal space via spatial Fourier analysis of images collected in the real space. We describe the main features of such digital Fourier microscopy (DFM), by providing examples of several possible experimental implementations of it, some of which not yet realized in practice. We also provide an overview of experimental results obtained with DFM for the study of the dynamics of soft materials. Finally, we outline possible future developments of DFM that would ease its adoption as a standard laboratory method. (topical review)

Toward single cell traction microscopy within 3D collagen matrices

International Nuclear Information System (INIS)

Hall, Matthew S.; Long, Rong; Feng, Xinzeng; Huang, YuLing; Hui, Chung-Yuen; Wu, Mingming

2013-01-01

Mechanical interaction between the cell and its extracellular matrix (ECM) regulates cellular behaviors, including proliferation, differentiation, adhesion, and migration. Cells require the three-dimensional (3D) architectural support of the ECM to perform physiologically realistic functions. However, current understanding of cell–ECM and cell–cell mechanical interactions is largely derived from 2D cell traction force microscopy, in which cells are cultured on a flat substrate. 3D cell traction microscopy is emerging for mapping traction fields of single animal cells embedded in either synthetic or natively derived fibrous gels. We discuss here the development of 3D cell traction microscopy, its current limitations, and perspectives on the future of this technology. Emphasis is placed on strategies for applying 3D cell traction microscopy to individual tumor cell migration within collagen gels. - Highlights: • Review of the current state of the art in 3D cell traction force microscopy. • Bulk and micro-characterization of remodelable fibrous collagen gels. • Strategies for performing 3D cell traction microscopy within collagen gels

Toward single cell traction microscopy within 3D collagen matrices

Energy Technology Data Exchange (ETDEWEB)

Hall, Matthew S. [Department of Biological and Environmental Engineering, Cornell University, Ithaca, NY 14853 (United States); Long, Rong [Department of Mechanical Engineering, University of Alberta, Edmonton, AB, Canada T6G 2G8 (Canada); Feng, Xinzeng [Department of Mechanical and Aerospace Engineering, Cornell University, Ithaca, NY 14853 (United States); Huang, YuLing [Department of Biological and Environmental Engineering, Cornell University, Ithaca, NY 14853 (United States); Hui, Chung-Yuen [Department of Mechanical and Aerospace Engineering, Cornell University, Ithaca, NY 14853 (United States); Wu, Mingming, E-mail: mw272@cornell.edu [Department of Biological and Environmental Engineering, Cornell University, Ithaca, NY 14853 (United States)

2013-10-01

Mechanical interaction between the cell and its extracellular matrix (ECM) regulates cellular behaviors, including proliferation, differentiation, adhesion, and migration. Cells require the three-dimensional (3D) architectural support of the ECM to perform physiologically realistic functions. However, current understanding of cell–ECM and cell–cell mechanical interactions is largely derived from 2D cell traction force microscopy, in which cells are cultured on a flat substrate. 3D cell traction microscopy is emerging for mapping traction fields of single animal cells embedded in either synthetic or natively derived fibrous gels. We discuss here the development of 3D cell traction microscopy, its current limitations, and perspectives on the future of this technology. Emphasis is placed on strategies for applying 3D cell traction microscopy to individual tumor cell migration within collagen gels. - Highlights: • Review of the current state of the art in 3D cell traction force microscopy. • Bulk and micro-characterization of remodelable fibrous collagen gels. • Strategies for performing 3D cell traction microscopy within collagen gels.

Super-Resolution Microscopy: Shedding Light on the Cellular Plasma Membrane.

Science.gov (United States)

Stone, Matthew B; Shelby, Sarah A; Veatch, Sarah L

2017-06-14

Lipids and the membranes they form are fundamental building blocks of cellular life, and their geometry and chemical properties distinguish membranes from other cellular environments. Collective processes occurring within membranes strongly impact cellular behavior and biochemistry, and understanding these processes presents unique challenges due to the often complex and myriad interactions between membrane components. Super-resolution microscopy offers a significant gain in resolution over traditional optical microscopy, enabling the localization of individual molecules even in densely labeled samples and in cellular and tissue environments. These microscopy techniques have been used to examine the organization and dynamics of plasma membrane components, providing insight into the fundamental interactions that determine membrane functions. Here, we broadly introduce the structure and organization of the mammalian plasma membrane and review recent applications of super-resolution microscopy to the study of membranes. We then highlight some inherent challenges faced when using super-resolution microscopy to study membranes, and we discuss recent technical advancements that promise further improvements to super-resolution microscopy and its application to the plasma membrane.

Fine surface structure of unfixed and hydrated macrophages observed by laser-plasma x-ray contact microscopy

International Nuclear Information System (INIS)

Yamamoto, Yoshimasa; Friedman, Herman; Yoshimura, Hideyuki; Kinjo, Yasuhito; Shioda, Seiji; Debari, Kazuhiro; Shinohara, Kunio; Rajyaguru, Jayshree; Richardson, Martin

2000-01-01

A compact, high-resolution, laser-plasma, x-ray contact microscope using a table-top Nd:glass laser system has been developed and utilized for the analysis of the surface structure of live macrophages. Fine fluffy surface structures of murine peritoneal macrophages, which were live, hydrolyzed and not sliced and stained, were observed by the x-ray microscope followed by analysis using an atomic force microscopy. In order to compare with other techniques, a scanning electron microscopy (SEM) was utilized to observe the surface structure of the macrophages. The SEM offered a fine whole cell image of the same macrophages, which were fixed and dehydrated, but the surfaces were ruffled and different from that of x-ray images. A standard light microscope was also utilized to observe the shape of live whole macrophages. Light microscopy showed some fluffy surface structures of the macrophages, but the resolution was too low to observe the fine structures. Thus, the findings of fine fluffy surface structures of macrophages by x-ray microscopy provide valuable information for studies of phagocytosis, cell spreading and adherence, which are dependent on the surface structure of macrophages. Furthermore, the present study also demonstrates the usefulness of x-ray microscopy for analysis of structures of living cells

Transmission Electron Microscopy Physics of Image Formation

CERN Document Server

Kohl, Helmut

2008-01-01

Transmission Electron Microscopy: Physics of Image Formation presents the theory of image and contrast formation, and the analytical modes in transmission electron microscopy. The principles of particle and wave optics of electrons are described. Electron-specimen interactions are discussed for evaluating the theory of scattering and phase contrast. Also discussed are the kinematical and dynamical theories of electron diffraction and their applications for crystal-structure analysis and imaging of lattices and their defects. X-ray microanalysis and electron energy-loss spectroscopy are treated as analytical methods. Specimen damage and contamination by electron irradiation limits the resolution for biological and some inorganic specimens. This fifth edition includes discussion of recent progress, especially in the area of aberration correction and energy filtering; moreover, the topics introduced in the fourth edition have been updated. Transmission Electron Microscopy: Physics of Image Formation is written f...

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles.

Science.gov (United States)

Killingsworth, Murray C; Bobryshev, Yuri V

2016-08-07

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

High-Throughput Light Sheet Microscopy for the Automated Live Imaging of Larval Zebrafish

Science.gov (United States)

Baker, Ryan; Logan, Savannah; Dudley, Christopher; Parthasarathy, Raghuveer

The zebrafish is a model organism with a variety of useful properties; it is small and optically transparent, it reproduces quickly, it is a vertebrate, and there are a large variety of transgenic animals available. Because of these properties, the zebrafish is well suited to study using a variety of optical technologies including light sheet fluorescence microscopy (LSFM), which provides high-resolution three-dimensional imaging over large fields of view. Research progress, however, is often not limited by optical techniques but instead by the number of samples one can examine over the course of an experiment, which in the case of light sheet imaging has so far been severely limited. Here we present an integrated fluidic circuit and microscope which provides rapid, automated imaging of zebrafish using several imaging modes, including LSFM, Hyperspectral Imaging, and Differential Interference Contrast Microscopy. Using this system, we show that we can increase our imaging throughput by a factor of 10 compared to previous techniques. We also show preliminary results visualizing zebrafish immune response, which is sensitive to gut microbiota composition, and which shows a strong variability between individuals that highlights the utility of high throughput imaging. National Science Foundation, Award No. DBI-1427957.

A study of phase separation in peptide-loaded HPMC films using T(zero)-modulated temperature DSC, atomic force microscopy, and scanning electron microscopy.

Science.gov (United States)

Hussain, Samana; Grandy, David B; Reading, Mike; Craig, Duncan Q M

2004-07-01

Despite the widespread use of drug-loaded polymeric systems, there is still considerable uncertainty with regard to the nature of the distribution of the drug within the polymer matrix. The aim of this investigation was to develop thermal and microscopic techniques whereby the miscibility and spatial distribution of a model peptide, cyclosporin A (CyA), in hydroxypropyl methylcellulose (HPMC) films may be studied. The new technique of T(zero)-modulated temperature differential scanning calorimetry (T(zero) MTDSC), scanning electron microscopy (SEM), and pulse force mode atomic force microscopy (PFM-AFM) were used in conjunction to study films prepared using a solvent evaporation process, with a solvent extraction study performed to elucidate the nature of the observed phases. T(zero) MTDSC studies showed glass transitions for both the HPMC and CycA, with the T(g) for the HPMC and CycA seen for the mixed systems. SEM showed two spherical phases of differing electron density. PFM-AFM also showed spheres of differing adhesion that increased in size on addition of drug. Pixel intensity analysis indicated that the smaller spheres corresponded to CycA. Exposure of the films to dichloromethane, in which CycA is soluble but HPMC is not, resulted in the presence of voids that corresponded well to the spheres suggested to correspond to the drug. It was concluded that the system had undergone extensive or complete phase separation, and that the thermal and microscopic techniques outlined above are an effective means by which this issue may be studied. Copyright 2004 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 93:1672-1681, 2004

The 'grey area' between small cell and non-small cell lung carcinomas. Light and electron microscopy versus clinical data in 14 cases

NARCIS (Netherlands)

Mooi, W. J.; van Zandwijk, N.; Dingemans, K. P.; Koolen, M. G.; Wagenvoort, C. A.

1986-01-01

We studied 14 lung tumours which on light microscopy had posed difficulties on classification as either small cell or non-small cell carcinomas. The light and electron microscopical features were compared with patient follow-up data. Electron microscopy showed neuroendocrine granules in 12 cases,

Imaging bacterial spores by soft-x-ray microscopy

International Nuclear Information System (INIS)

Stead, A.D.; Ford, T.W.; Judge, J.

1997-01-01

Bacterial spores are able to survive dehydration, but neither the physiological nor structural basis of this have been fully elucidated. Furthermore, once hydrated, spores often require activation before they will germinate. Several treatments can be used to activate spores, but in the case of Bacillus subtlis the most effective is heat treatment. The physiological mechanism associated with activation is also not understood, but some workers suggest that the loss of calcium from the spores may be critical. However, just prior to germination, the spores change from being phase bright to phase dark when viewed by light microscopy. Imaging spores by soft x-ray microscopy is possible without fixation. Thus, in contrast to electron microscopy, it is possible to compare the structure of dehydrated and hydrated spores in a manner not possible previously. A further advantage is that it is possible to monitor individual spores by phase contrast light microscopy immediately prior to imaging with soft x-rays; whereas, with both electron microscopy and biochemical studies, it is a population of spores being studied without knowledge of the phase characteristics of individual spores. This study has therefore tried to compare dehydrated and hydrated spores and to determine if there is a mass loss from individual spores as they pass the transition from being phase bright to phase dark

Imaging bacterial spores by soft-x-ray microscopy

Energy Technology Data Exchange (ETDEWEB)

Stead, A.D.; Ford, T.W. [Univ. of London, Surrey (United Kingdom); Judge, J. [Unilever plc, Sharnbrook (United Kingdom)] [and others

1997-04-01

Bacterial spores are able to survive dehydration, but neither the physiological nor structural basis of this have been fully elucidated. Furthermore, once hydrated, spores often require activation before they will germinate. Several treatments can be used to activate spores, but in the case of Bacillus subtlis the most effective is heat treatment. The physiological mechanism associated with activation is also not understood, but some workers suggest that the loss of calcium from the spores may be critical. However, just prior to germination, the spores change from being phase bright to phase dark when viewed by light microscopy. Imaging spores by soft x-ray microscopy is possible without fixation. Thus, in contrast to electron microscopy, it is possible to compare the structure of dehydrated and hydrated spores in a manner not possible previously. A further advantage is that it is possible to monitor individual spores by phase contrast light microscopy immediately prior to imaging with soft x-rays; whereas, with both electron microscopy and biochemical studies, it is a population of spores being studied without knowledge of the phase characteristics of individual spores. This study has therefore tried to compare dehydrated and hydrated spores and to determine if there is a mass loss from individual spores as they pass the transition from being phase bright to phase dark.

Electron microscopy of nuclear zirconium alloys

International Nuclear Information System (INIS)

Versaci, R.A.; Ipohorski, Miguel

1986-01-01

Transmission electron microscopy observations of the microstructure of zirconium alloys used in fuel sheaths of nuclear power reactors are reported. Specimens were observed after different thermal and mechanical treatment, similar to those actually used during fabrication of the sheaths. Electron micrographs and electron diffraction patterns of second phase particles present in zircaloy-2 and zircaloy-4 were also obtained, as well as some characteristic parameters. Images of oxides and hydrides most commonly present in zirconium alloys are also shown. Finally, the structure of a Zr-2,5Nb alloy used in CANDU reactors pressure tubes, is observed by electron microscopy. (Author) [es

Two-Photon Excitation Microscopy for the Study of Living Cells and Tissues

Science.gov (United States)

Benninger, Richard K.P.; Piston, David W.

2013-01-01

Two-photon excitation microscopy is an alternative to confocal microscopy that provides advantages for three-dimensional and deep tissue imaging. This unit will describe the basic physical principles behind two-photon excitation and discuss the advantages and limitations of its use in laser-scanning microscopy. The principal advantages of two-photon microscopy are reduced phototoxicity, increased imaging depth, and the ability to initiate highly localized photochemistry in thick samples. Practical considerations for the application of two-photon microscopy will then be discussed, including recent technological advances. This unit will conclude with some recent applications of two-photon microscopy that highlight the key advantages over confocal microscopy and the types of experiments which would benefit most from its application. PMID:23728746

On some aspects of high voltage electron microscopy

International Nuclear Information System (INIS)

Jouffrey, B.; Trinquier, J.

1987-01-01

The present paper deals with high voltage electron microscopy (HVEM). It is an overview on this domain due to the pionneer work of G. Dupouy which has permitted to perform a new kind of electron microscopy. Since this time, HVEM has shown its interest in high resolution, irradiations, chemical analysis, in situ experiments

Virtual Hematoxylin and Eosin Transillumination Microscopy Using Epi-Fluorescence Imaging.

Directory of Open Access Journals (Sweden)

Michael G Giacomelli

Full Text Available We derive a physically realistic model for the generation of virtual transillumination, white light microscopy images using epi-fluorescence measurements from thick, unsectioned tissue. We demonstrate this technique by generating virtual transillumination H&E images of unsectioned human breast tissue from epi-fluorescence multiphoton microscopy data. The virtual transillumination algorithm is shown to enable improved contrast and color accuracy compared with previous color mapping methods. Finally, we present an open source implementation of the algorithm in OpenGL, enabling real-time GPU-based generation of virtual transillumination microscopy images using conventional fluorescence microscopy systems.

Confocal fluorescence microscopy in a murine model of microdissection testicular sperm extraction to improve sperm retrieval.

Science.gov (United States)

Smith, Ryan P; Lowe, Greg J; Kavoussi, Parviz K; Steers, William D; Costabile, Raymond A; Herr, John C; Shetty, Jagathpala; Lysiak, Jeffrey J

2012-05-01

Microdissection testicular sperm extraction markedly improves the sperm retrieval rates in men with nonobstructive azoospermia. However, localizing sperm foci can be time-consuming and it is not always successful. Fiberoptic confocal fluorescent microscopy offers the advantage of rapid in vivo detection of fluorescently labeled sperm in the seminiferous tubules. After establishing the feasibility of fiberoptic confocal fluorescent microscopy to identify antibody labeled sperm in vivo C57/B6 mice underwent intraperitoneal injection of busulfan to induce azoospermia. During spermatogenesis reestablishment at approximately 16 weeks the mice were anesthetized and the testes were delivered through a low midline incision. Fluorescein isothiocyanate labeled antibody to intra-acrosomal protein Hs-14 was injected retrograde into a single murine rete testis. The testes were imaged in vivo with fiberoptic confocal fluorescent microscopy and sperm foci were detected. The respective seminiferous tubules were excised and squash prepared for immunofluorescence microscopy. Sperm foci were identified in the testis injected with fluorescently tagged antibody by in vivo fiberoptic confocal fluorescence microscopy. The contralateral control testis of each mouse showed no specific signal. Immunofluorescence microscopy of the excised tubules provided morphological confirmation of the presence of labeled sperm with an absence in controls. Findings were consistent in the feasibility portion of the study and in the busulfan model of nonobstructive azoospermia. Fiberoptic confocal fluorescent microscopy was feasible during microdissection testicular sperm extraction in an azoospermic mouse model to identify fluorescently labeled sperm in vivo. Translation to the clinical setting could decrease operative time and improve the sperm harvest rate. Copyright © 2012 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Electron microscopy study of microbial mat in the North Fiji basin hydrothermal vent

Science.gov (United States)

Park, H.; Kim, J. W.; Lee, J. W.

2017-12-01

Hydrothermal vent systems consisting of hydrothermal vent, hydrothermal sediment and microbial mat are widely spread around the ocean, particularly spreading axis, continental margin and back-arc basin. Scientists have perceived that the hydrothermal systems, which reflect the primeval earth environment, are one of the best places to reveal the origin of life and extensive biogeochemical process of microbe-mineral interaction. In the present study multiline of analytical methods (X-Ray Diffraction (XRD), Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM)) were utilized to investigate the mineralogy/chemistry of microbe-mineral interaction in hydrothermal microbial mat. Microbial mat samples were recovered by Canadian scientific submersible ROPOS on South Pacific North Fiji basin KIOST hydrothermal vent expedition 1602. XRD analysis showed that red-colored microbial mat contains Fe-oxides and Fe-oxyhydroxides. Various morphologies of minerals in the red-colored microbial mat observed by SEM are mainly showed sheath shaped, resembled with Leptothrix microbial structure, stalks shaped, similar with Marioprofundus microbial structure and globule shaped microbial structures. They are also detected with DNA analysis. The cross sectional observation of microbial structures encrusted with Fe-oxide and Fe-oxyhydroxide at a nano scale by Transmission Electron Microscopy (TEM) and Focused Ion Beam (FIB) technique was developed to verify the structural/biogeochemical properties in the microbe-mineral interaction. Systematic nano-scale measurements on the biomineralization in the microbial mat leads the understandings of biogeochemical environments around the hydrothermal vent.

Scanning Near-Field Optical Microscopy

Directory of Open Access Journals (Sweden)

Dušan Vobornik

2008-02-01

Full Text Available An average human eye can see details down to 0,07 mm in size. The ability to see smaller details of the matter is correlated with the development of the science and the comprehension of the nature. Today’s science needs eyes for the nano-world. Examples are easily found in biology and medical sciences. There is a great need to determine shape, size, chemical composition, molecular structure and dynamic properties of nano-structures. To do this, microscopes with high spatial, spectral and temporal resolution are required. Scanning Near-field Optical Microscopy (SNOM is a new step in the evolution of microscopy. The conventional, lens-based microscopes have their resolution limited by diffraction. SNOM is not subject to this limitation and can offer up to 70 times better resolution.

Scanning near-field optical microscopy.

Science.gov (United States)

Vobornik, Dusan; Vobornik, Slavenka

2008-02-01

An average human eye can see details down to 0,07 mm in size. The ability to see smaller details of the matter is correlated with the development of the science and the comprehension of the nature. Today's science needs eyes for the nano-world. Examples are easily found in biology and medical sciences. There is a great need to determine shape, size, chemical composition, molecular structure and dynamic properties of nano-structures. To do this, microscopes with high spatial, spectral and temporal resolution are required. Scanning Near-field Optical Microscopy (SNOM) is a new step in the evolution of microscopy. The conventional, lens-based microscopes have their resolution limited by diffraction. SNOM is not subject to this limitation and can offer up to 70 times better resolution.

Selective sensitivity in Kerr microscopy.

Science.gov (United States)

Soldatov, I V; Schäfer, R

2017-07-01

A new technique for contrast separation in wide-field magneto-optical Kerr microscopy is introduced. Utilizing the light from eight light emitting diodes, guided to the microscope by glass fibers and being switched synchronously with the camera exposure, domain images with orthogonal in-plane sensitivity can be displayed simultaneously at real-time, and images with pure in-plane or polar contrast can be obtained. The benefit of this new method of contrast separation is demonstrated for Permalloy films, a NdFeB sinter magnet, and a cobalt crystal. Moreover, the new technique is shown to strongly enhance the sensitivity of Kerr microscopy by eliminating parasitic contrast contributions occurring in conventional setups. A doubling of the in-plane domain contrast and a sensitivity to Kerr rotations as low as 0.6 mdeg is demonstrated.

Selective sensitivity in Kerr microscopy

Science.gov (United States)

Soldatov, I. V.; Schäfer, R.

2017-07-01

A new technique for contrast separation in wide-field magneto-optical Kerr microscopy is introduced. Utilizing the light from eight light emitting diodes, guided to the microscope by glass fibers and being switched synchronously with the camera exposure, domain images with orthogonal in-plane sensitivity can be displayed simultaneously at real-time, and images with pure in-plane or polar contrast can be obtained. The benefit of this new method of contrast separation is demonstrated for Permalloy films, a NdFeB sinter magnet, and a cobalt crystal. Moreover, the new technique is shown to strongly enhance the sensitivity of Kerr microscopy by eliminating parasitic contrast contributions occurring in conventional setups. A doubling of the in-plane domain contrast and a sensitivity to Kerr rotations as low as 0.6 mdeg is demonstrated.

Lobster eye as a collector for water window microscopy

Science.gov (United States)

Pina, L.; Maršíková, V.; Inneman, A.; Nawaz, M. F.; Jančárek, A.; Havlíková, R.

2017-08-01

Imaging in EUV, SXR and XR spectral bands of radiation is of increasing interest. Material science, biology and hot plasma are examples of relevant fast developing areas. Applications include spectroscopy, astrophysics, Soft X-ray Ray metrology, Water Window microscopy, radiography and tomography. Especially Water Window imaging has still not fully recognized potential in biology and medicine microscopy applications. Theoretical study and design of Lobster Eye (LE) optics as a collector for water window (WW) microscopy and comparison with a similar size ellipsoidal mirror condensor are presented.

Quantitative imaging of lipids in live mouse oocytes and early embryos using CARS microscopy

Science.gov (United States)

Bradley, Josephine; Pope, Iestyn; Masia, Francesco; Sanusi, Randa; Langbein, Wolfgang; Borri, Paola

2016-01-01

Mammalian oocytes contain lipid droplets that are a store of fatty acids, whose metabolism plays a substantial role in pre-implantation development. Fluorescent staining has previously been used to image lipid droplets in mammalian oocytes and embryos, but this method is not quantitative and often incompatible with live cell imaging and subsequent development. Here we have applied chemically specific, label-free coherent anti-Stokes Raman scattering (CARS) microscopy to mouse oocytes and pre-implantation embryos. We show that CARS imaging can quantify the size, number and spatial distribution of lipid droplets in living mouse oocytes and embryos up to the blastocyst stage. Notably, it can be used in a way that does not compromise oocyte maturation or embryo development. We have also correlated CARS with two-photon fluorescence microscopy simultaneously acquired using fluorescent lipid probes on fixed samples, and found only a partial degree of correlation, depending on the lipid probe, clearly exemplifying the limitation of lipid labelling. In addition, we show that differences in the chemical composition of lipid droplets in living oocytes matured in media supplemented with different saturated and unsaturated fatty acids can be detected using CARS hyperspectral imaging. These results demonstrate that CARS microscopy provides a novel non-invasive method of quantifying lipid content, type and spatial distribution with sub-micron resolution in living mammalian oocytes and embryos. PMID:27151947

Effects of phase change on reflection in phase-measuring interference microscopy

OpenAIRE

Dubois , Arnaud

2004-01-01

International audience; We show by analytical and numerical calculations that the phase change on reflection that occurs in interference microscopy is almost independent of the numerical aperture of the objective. The shift of the microscope interferogram response due to the phase change on reflection, however, increases with the numerical aperture. Measurements of the interferogram shift are made with a Linnik interference microscope equipped with various numerical-aperture objectives and ar...

Two-dimensional dopant profiling by electrostatic force microscopy using carbon nanotube modified cantilevers

International Nuclear Information System (INIS)

Chin, S.-C.; Chang, Y.-C.; Chang, C.-S.; Tsong, T T; Hsu, Chen-Chih; Wu, Chih-I; Lin, W-H; Woon, W-Y; Lin, L-T; Tao, H-J

2008-01-01

A two-dimensional (2D) dopant profiling technique is demonstrated in this work. We apply a unique cantilever probe in electrostatic force microscopy (EFM) modified by the attachment of a multiwalled carbon nanotube (MWNT). Furthermore, the tip apex of the MWNT was trimmed to the sharpness of a single-walled carbon nanotube (SWNT). This ultra-sharp MWNT tip helps us to resolve dopant features to within 10 nm in air, which approaches the resolution achieved by ultra-high vacuum scanning tunnelling microscopy (UHV STM). In this study, the CNT-probed EFM is used to profile 2D buried dopant distribution under a nano-scale device structure and shows the feasibility of device characterization for sub-45 nm complementary metal-oxide-semiconductor (CMOS) field-effect transistors

Sample preparation method for scanning force microscopy

CERN Document Server

Jankov, I R; Szente, R N; Carreno, M N P; Swart, J W; Landers, R

2001-01-01

We present a method of sample preparation for studies of ion implantation on metal surfaces. The method, employing a mechanical mask, is specially adapted for samples analysed by Scanning Force Microscopy. It was successfully tested on polycrystalline copper substrates implanted with phosphorus ions at an acceleration voltage of 39 keV. The changes of the electrical properties of the surface were measured by Kelvin Probe Force Microscopy and the surface composition was analysed by Auger Electron Spectroscopy.

Three-dimensional live microscopy beyond the diffraction limit

International Nuclear Information System (INIS)

Fiolka, Reto

2013-01-01

In fluorescence microscopy it has become possible to fundamentally overcome the diffraction limited resolution in all three spatial dimensions. However, to have the most impact in biological sciences, new optical microscopy techniques need to be compatible with live cell imaging: image acquisition has to be fast enough to capture cellular dynamics at the new resolution limit while light exposure needs to be minimized to prevent photo-toxic effects. With increasing spatial resolution, these requirements become more difficult to meet, even more so when volumetric imaging is performed. In this review, techniques that have been successfully applied to three-dimensional, super-resolution live microscopy are presented and their relative strengths and weaknesses are discussed. (special issue article)

The mechanism of borax crystallization using in situ optical microscopy and AFM

International Nuclear Information System (INIS)

Suharso, G.; Parkinson, M.; Ogden, M.

2002-01-01

Full text: The quality of high-purity borax depends both on the concentrations of the impurities and the product appearance, which are mainly determined by the size and morphology of the crystals. Thus, knowledge about crystallization of borax is of direct relevance to the industrial production of borax. In addition, fundamental studies of borax crystallization will provide results of relevance to the crystallization of other economically important materials. An investigation into the fundamental mechanism of crystal growth of borax from aqueous solution was carried out, as a model system. The investigation focussed on the growth mechanism, and the influence of factors such as solution supersaturation, temperature, crystal size and solution flow on the rate of crystal growth. In situ optical microscopy was used to determine growth rates of three different faces of borax crystals at 20, 25, 30, and 35 deg C, at various concentrations. It was found that the growth rate increases with increasing temperature and supersaturation. At low concentration , growth on the (010), (001), and (111) faces occurs via a spiral growth mechanism and at high concentration birth and spread is the principal mechanism operating. The activation energy for the different mechanisms was determined. Examination by ex situ Atomic Force Microscopy (AFM) showed features suggesting that the (100), (010), (001) faces of borax crystals grow by spiral mechanism at low concentration and two dimensional nucleation at high concentration. These experiments support the data obtained from in situ optical microscopy. Copyright (2002) Australian Society for Electron Microscopy Inc

Using environmental forensic microscopy in exposure science.

Science.gov (United States)

Millette, James R; Brown, Richard S; Hill, Whitney B

2008-01-01

Environmental forensic microscopy investigations are based on the methods and procedures developed in the fields of criminal forensics, industrial hygiene and environmental monitoring. Using a variety of microscopes and techniques, the environmental forensic scientist attempts to reconstruct the sources and the extent of exposure based on the physical evidence left behind after particles are exchanged between an individual and the environments he or she passes through. This article describes how environmental forensic microscopy uses procedures developed for environmental monitoring, criminal forensics and industrial hygiene investigations. It provides key references to the interdisciplinary approach used in microscopic investigations. Case studies dealing with lead, asbestos, glass fibers and other particulate contaminants are used to illustrate how environmental forensic microscopy can be very useful in the initial stages of a variety of environmental exposure characterization efforts to eliminate some agents of concern and to narrow the field of possible sources of exposure.

3DSEM: A 3D microscopy dataset

Directory of Open Access Journals (Sweden)

Ahmad P. Tafti

2016-03-01

Full Text Available The Scanning Electron Microscope (SEM as a 2D imaging instrument has been widely used in many scientific disciplines including biological, mechanical, and materials sciences to determine the surface attributes of microscopic objects. However the SEM micrographs still remain 2D images. To effectively measure and visualize the surface properties, we need to truly restore the 3D shape model from 2D SEM images. Having 3D surfaces would provide anatomic shape of micro-samples which allows for quantitative measurements and informative visualization of the specimens being investigated. The 3DSEM is a dataset for 3D microscopy vision which is freely available at [1] for any academic, educational, and research purposes. The dataset includes both 2D images and 3D reconstructed surfaces of several real microscopic samples. Keywords: 3D microscopy dataset, 3D microscopy vision, 3D SEM surface reconstruction, Scanning Electron Microscope (SEM

Immunochemical localization of ribulose-1,5-bisphosphate carboxylase in the symbiont-containing gills of Solemya velum (Bivalvia: Mollusca).

Science.gov (United States)

Cavanaugh, C M; Abbott, M S; Veenhuis, M

1988-10-01

The distribution of the Calvin cycle enzyme ribulose-1,5-bisphosphate carboxylase (RbuP(2)Case; EC 4.1.1.39) was examined by using two immunological methods in tissues of Solemya velum, an Atlantic coast bivalve containing putative chemoautotrophic symbionts. Antibodies elicited by the purified large subunit of RbuP(2)Case from tobacco (Nicotiana tabacum) cross-reacted on immunoblots with a protein of similar molecular mass occurring in extracts of the symbiont-containing gill tissue of S. velum. No cross-reactivity was detected in symbiont-free tissue extracts. The antiserum also cross-reacted in immunoblots with proteins of Thiobacillus neapolitanus, a free-living sulfuroxidizing chemoautotroph whose RbuP(2)Case has been well characterized. In protein A-gold immunoelectron microscopy studies, this antiserum consistently labeled the symbionts but not surrounding host gill tissue, indicating that the symbionts are responsible for the RbuP(2)Case activity.

GLUT4 in cultured skeletal myotubes is segregated from the transferrin receptor and stored in vesicles associated with TGN

DEFF Research Database (Denmark)

Ralston, E; Ploug, Thorkil

1996-01-01

of the constitutive endosomal-lysosomal pathway. To address this question, we have investigated the localization of the endogenous GLUT4 in non-stimulated skeletal myotubes from the cell line C2, by immunofluorescence and immunoelectron microscopy. We have used a panel of antibodies to markers of the Golgi complex...... and in vesicles just beyond, i.e. in the structures that constitute the trans-Golgi network (TGN). In myotubes treated with brefeldin A, the immunofluorescence pattern of GLUT4 is modified, but it differs from both Golgi complex markers and TGN38. Instead, it resembles the pattern of the transferrin receptor...... to the GLUT4-containing tubulo-vesicular elements. In brefeldin A-treated cells, a network of tubules of approximately 70 nm diameter, studded with varicosities, stains for both GLUT4 and transferrin receptor, suggesting that brefeldin A has caused fusion of the transferrin receptor and GLUT4-containing...

Improved axial resolution of FINCH fluorescence microscopy when combined with spinning disk confocal microscopy.

Science.gov (United States)

Siegel, Nisan; Brooker, Gary

2014-09-22

FINCH holographic fluorescence microscopy creates super-resolved images with enhanced depth of focus. Addition of a Nipkow disk real-time confocal image scanner is shown to reduce the FINCH depth of focus while improving transverse confocal resolution in a combined method called "CINCH".

Influence of Oxygen Pressure on the Domain Dynamics and Local Electrical Properties of BiFe0.95Mn0.05O3 Thin Films Studied by Piezoresponse Force Microscopy and Conductive Atomic Force Microscopy

Directory of Open Access Journals (Sweden)

Kunyu Zhao

2017-11-01

Full Text Available In this work, we have studied the microstructures, nanodomains, polarization preservation behaviors, and electrical properties of BiFe0.95Mn0.05O3 (BFMO multiferroic thin films, which have been epitaxially created on the substrates of SrRuO3, SrTiO3, and TiN-buffered (001-oriented Si at different oxygen pressures via piezoresponse force microscopy and conductive atomic force microscopy. We found that the pure phase state, inhomogeneous piezoresponse force microscopy (PFM response, low leakage current with unidirectional diode-like properties, and orientation-dependent polarization reversal properties were found in BFMO thin films deposited at low oxygen pressure. Meanwhile, these films under high oxygen pressures resulted in impurities in the secondary phase in BFMO films, which caused a greater leakage that hindered the polarization preservation capability. Thus, this shows the important impact of the oxygen pressure on modulating the physical effects of BFMO films.

Fluorescence confocal polarizing microscopy

Indian Academy of Sciences (India)

Much of the modern understanding of orientational order in liquid crystals (LCs) is based on polarizing microscopy (PM). A PM image bears only two-dimensional (2D) information, integrating the 3D pattern of optical birefringence over the path of light. Recently, we proposed a technique to image 3D director patterns by ...

High-speed atomic force microscopy combined with inverted optical microscopy for studying cellular events.

Science.gov (United States)

Suzuki, Yuki; Sakai, Nobuaki; Yoshida, Aiko; Uekusa, Yoshitsugu; Yagi, Akira; Imaoka, Yuka; Ito, Shuichi; Karaki, Koichi; Takeyasu, Kunio

2013-01-01

A hybrid atomic force microscopy (AFM)-optical fluorescence microscopy is a powerful tool for investigating cellular morphologies and events. However, the slow data acquisition rates of the conventional AFM unit of the hybrid system limit the visualization of structural changes during cellular events. Therefore, high-speed AFM units equipped with an optical/fluorescence detection device have been a long-standing wish. Here we describe the implementation of high-speed AFM coupled with an optical fluorescence microscope. This was accomplished by developing a tip-scanning system, instead of a sample-scanning system, which operates on an inverted optical microscope. This novel device enabled the acquisition of high-speed AFM images of morphological changes in individual cells. Using this instrument, we conducted structural studies of living HeLa and 3T3 fibroblast cell surfaces. The improved time resolution allowed us to image dynamic cellular events.

Calcite biomineralization in coccoliths: Evidence from atomic force microscopy (AFM)

DEFF Research Database (Denmark)

Henriksen, Karen; Stipp, S.L.S.

2002-01-01

geochemistry, crystal orientation, coccolith function, biomineralization, biological calcite, atomic force microscopy......geochemistry, crystal orientation, coccolith function, biomineralization, biological calcite, atomic force microscopy...

Stereoscopic and photometric surface reconstruction in scanning electron microscopy

International Nuclear Information System (INIS)

Scherer, S.

2000-01-01

The scanning electron microscope (SEM) is one of the most important devices to examine microscopic structures as it offers images of a high contrast range with a large depth of focus. Nevertheless, three-dimensional measurements, as desired in fracture mechanics, have previously not been accomplished. This work presents a system for automatic, robust and dense surface reconstruction in scanning electron microscopy combining new approaches in shape from stereo and shape from photometric stereo. The basic theoretical assumption for a known adaptive window algorithm is shown not to hold in scanning electron microscopy. A constraint derived from this observation yields a new, simplified, hence faster calculation of the adaptive window. The correlation measure itself is obtained by a new ordinal measure coefficient. Shape from photometric stereo in the SEM is formulated by relating the image formation process with conventional photography. An iterative photometric ratio reconstruction is invented based on photometric ratios of backscatter electron images. The performance of the proposed system is evaluated using ground truth data obtained by three alternative shape recovery devices. Most experiments showed relative height accuracy within the tolerances of the alternative devices. (author)

Correlative Analysis of Immunoreactivity in Confocal Laser-Scanning Microscopy and Scanning Electron Microscopy with Focused Ion Beam Milling

Directory of Open Access Journals (Sweden)

Takahiro eSonomura

2013-02-01

Full Text Available Three-dimensional reconstruction of ultrastructure of rat brain with minimal effort has recently been realized by scanning electron microscopy combined with focused ion beam milling (FIB-SEM. Because application of immunohistochemical staining to electron microscopy has a great advantage in that molecules of interest are specifically localized in ultrastructures, we here tried to apply immunocytochemistry to FIB-SEM and correlate immunoreactivity in confocal laser-scanning microcopy (CF-LSM with that in FIB-SEM. The dendrites of medium-sized spiny neurons in rat neostriatum were visualized with a recombinant viral vector, which labeled the infected neurons with membrane-targeted GFP in a Golgi stain-like fashion, and thalamostriatal afferent terminals were immunolabeled with Cy5 fluorescence for vesicular glutamate transporter 2 (VGluT2. After detecting the sites of terminals apposed to the dendrites in CF-LSM, GFP and VGluT2 immunoreactivities were further developed for electron microscopy by the immunogold/silver enhancement and immunoperoxidase/diaminobenzidine (DAB methods, respectively. In the contrast-inverted FIB-SEM images, silver precipitation and DAB deposits were observed as fine dark grains and diffuse dense profiles, respectively, indicating that these immunoreactivities were easily recognizable as in the images of transmission electron microscopy. In the sites of interest, some appositions were revealed to display synaptic specialization of asymmetric type. The present method is thus useful in the three-dimensional analysis of immunocytochemically differentiated synaptic connection in the central neural circuit.

Coherent Raman Imaging of Live Muscle Sarcomeres Assisted by SFG Microscopy.

Science.gov (United States)

Kim, Hyunmin; Kim, Do-Young; Joo, Kyung-Il; Kim, Jung-Hye; Jeong, Soon Moon; Lee, Eun Seong; Hahm, Jeong-Hoon; Kim, Kyuhyung; Moon, Dae Woon

2017-08-23

In this study, we used spectrally focused coherent anti-Stokes Raman scattering (spCARS) microscopy assisted by sum-frequency generation (SFG) to monitor the variations in the structural morphology and molecular vibrations of a live muscle of Caenorhabditis elegans. The subunits of the muscle sarcomeres, such as the M-line, myosin, dense body, and α-actinin, were alternatively observed using spCARS microscopy for different sample orientations, with the guidance of a myosin positional marker captured by SFG microscopy. Interestingly enough, the beam polarization dependence of the spCARS contrasts for two parallel subunits (dense body and myosin) showed a ~90° phase difference. The chemically sensitive spCARS spectra induced by the time-varying overlap of two pulses allowed (after a robust subtraction of the non-resonant background using a modified Kramers-Krönig transformation method) high-fidelity detection of various genetically modified muscle sarcomeres tuned to the C-H vibration (2800-3100 cm -1 ). Conversely, SFG image mapping assisted by phase-retrieved spCARS spectra also facilitated label-free monitoring of the changes in the muscle content of C. elegans that are associated with aging, based on the hypothesis that the C-H vibrational modes could serve as qualitative chemical markers sensitive to the amount and/or structural modulation of the muscle.

Clinical applications of corneal confocal microscopy

Directory of Open Access Journals (Sweden)

Mitra Tavakoli

2008-06-01

Full Text Available Mitra Tavakoli1, Parwez Hossain2, Rayaz A Malik11Division of Cardiovascular Medicine, University of Manchester and Manchester Royal Infirmary, Manchester, UK; 2University of Southampton, Southampton Eye Unit, Southampton General Hospital, Southampton, UKAbstract: Corneal confocal microscopy is a novel clinical technique for the study of corneal cellular structure. It provides images which are comparable to in-vitro histochemical techniques delineating corneal epithelium, Bowman’s layer, stroma, Descemet’s membrane and the corneal endothelium. Because, corneal confocal microscopy is a non invasive technique for in vivo imaging of the living cornea it has huge clinical potential to investigate numerous corneal diseases. Thus far it has been used in the detection and management of pathologic and infectious conditions, corneal dystrophies and ecstasies, monitoring contact lens induced corneal changes and for pre and post surgical evaluation (PRK, LASIK and LASEK, flap evaluations and Radial Keratotomy, and penetrating keratoplasty. Most recently it has been used as a surrogate for peripheral nerve damage in a variety of peripheral neuropathies and may have potential in acting as a surrogate marker for endothelial abnormalities.Keywords: corneal confocal microscopy, cornea, infective keratitis, corneal dystrophy, neuropathy

Restoration of uneven illumination in light sheet microscopy images.

Science.gov (United States)

Uddin, Mohammad Shorif; Lee, Hwee Kuan; Preibisch, Stephan; Tomancak, Pavel

2011-08-01

Light microscopy images suffer from poor contrast due to light absorption and scattering by the media. The resulting decay in contrast varies exponentially across the image along the incident light path. Classical space invariant deconvolution approaches, while very effective in deblurring, are not designed for the restoration of uneven illumination in microscopy images. In this article, we present a modified radiative transfer theory approach to solve the contrast degradation problem of light sheet microscopy (LSM) images. We confirmed the effectiveness of our approach through simulation as well as real LSM images.

A dark mode in scanning thermal microscopy

Science.gov (United States)

Ramiandrisoa, Liana; Allard, Alexandre; Joumani, Youssef; Hay, Bruno; Gomés, Séverine

2017-12-01

The need for high lateral spatial resolution in thermal science using Scanning Thermal Microscopy (SThM) has pushed researchers to look for more and more tiny probes. SThM probes have consequently become more and more sensitive to the size effects that occur within the probe, the sample, and their interaction. Reducing the tip furthermore induces very small heat flux exchanged between the probe and the sample. The measurement of this flux, which is exploited to characterize the sample thermal properties, requires then an accurate thermal management of the probe-sample system and to reduce any phenomenon parasitic to this system. Classical experimental methodologies must then be constantly questioned to hope for relevant and interpretable results. In this paper, we demonstrate and estimate the influence of the laser of the optical force detection system used in the common SThM setup that is based on atomic-force microscopy equipment on SThM measurements. We highlight the bias induced by the overheating due to the laser illumination on the measurements performed by thermoresistive probes (palladium probe from Kelvin Nanotechnology). To face this issue, we propose a new experimental procedure based on a metrological approach of the measurement: a SThM "dark mode." The comparison with the classical procedure using the laser shows that errors between 14% and 37% can be reached on the experimental data exploited to determine the heat flux transferred from the hot probe to the sample.

Molecular Composition and Ultrastructure of the Caveolar Coat Complex

Science.gov (United States)

Ludwig, Alexander; Howard, Gillian; Mendoza-Topaz, Carolina; Deerinck, Thomas; Mackey, Mason; Sandin, Sara; Ellisman, Mark H.; Nichols, Benjamin J.

2013-01-01

Caveolae are an abundant feature of the plasma membrane of many mammalian cell types, and have key roles in mechano-transduction, metabolic regulation, and vascular permeability. Caveolin and cavin proteins, as well as EHD2 and pacsin 2, are all present in caveolae. How these proteins assemble to form a protein interaction network for caveolar morphogenesis is not known. Using in vivo crosslinking, velocity gradient centrifugation, immuno-isolation, and tandem mass spectrometry, we determine that cavins and caveolins assemble into a homogenous 80S complex, which we term the caveolar coat complex. There are no further abundant components within this complex, and the complex excludes EHD2 and pacsin 2. Cavin 1 forms trimers and interacts with caveolin 1 with a molar ratio of about 1∶4. Cavins 2 and 3 compete for binding sites within the overall coat complex, and form distinct subcomplexes with cavin 1. The core interactions between caveolin 1 and cavin 1 are independent of cavin 2, cavin 3, and EHD2 expression, and the cavins themselves can still interact in the absence of caveolin 1. Using immuno-electron microscopy as well as a recently developed protein tag for electron microscopy (MiniSOG), we demonstrate that caveolar coat complexes form a distinct coat all around the caveolar bulb. In contrast, and consistent with our biochemical data, EHD2 defines a different domain at the caveolar neck. 3D electron tomograms of the caveolar coat, labeled using cavin-MiniSOG, show that the caveolar coat is composed of repeating units of a unitary caveolar coat complex. PMID:24013648

Fidelity imaging for atomic force microscopy

Energy Technology Data Exchange (ETDEWEB)

Ghosal, Sayan, E-mail: ghos0087@umn.edu; Salapaka, Murti, E-mail: murtis@umn.edu [Nanodynamics Systems Laboratory, Department of Electrical and Computer Engineering, University of Minnesota, Minneapolis, Minnesota 55455 (United States)

2015-01-05

Atomic force microscopy is widely employed for imaging material at the nanoscale. However, real-time measures on image reliability are lacking in contemporary atomic force microscopy literature. In this article, we present a real-time technique that provides an image of fidelity for a high bandwidth dynamic mode imaging scheme. The fidelity images define channels that allow the user to have additional authority over the choice of decision threshold that facilitates where the emphasis is desired, on discovering most true features on the sample with the possible detection of high number of false features, or emphasizing minimizing instances of false detections. Simulation and experimental results demonstrate the effectiveness of fidelity imaging.

Active Pixel Sensors for electron microscopy

Energy Technology Data Exchange (ETDEWEB)

Denes, P. [Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States)], E-mail: pdenes@lbl.gov; Bussat, J.-M. [Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States); Lee, Z.; Radmillovic, V. [National Center for Electron Microscopy, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States)

2007-09-01

The technology used for monolithic CMOS imagers, popular for cell phone cameras and other photographic applications, has been explored for charged particle tracking by the high-energy physics community for several years. This technology also lends itself to certain imaging detector applications in electron microscopy. We have been developing such detectors for several years at Lawrence Berkeley National Laboratory, and we and others have shown that this technology can offer excellent point-spread function, direct detection and high readout speed. In this paper, we describe some of the design constraints peculiar to electron microscopy and summarize where such detectors could play a useful role.

Transfer doping of single isolated nanodiamonds, studied by scanning probe microscopy techniques.

Science.gov (United States)

Bolker, Asaf; Saguy, Cecile; Kalish, Rafi

2014-09-26

The transfer doping of diamond surfaces has been applied in various novel two-dimensional electronic devices. Its extension to nanodiamonds (ND) is essential for ND-based applications in many fields. In particular, understanding the influence of the crystallite size on transfer doping is desirable. Here, we report the results of a detailed study of the electronic energetic band structure of single, isolated transfer-doped nanodiamonds with nanometric resolution using a combination of scanning tunneling spectroscopy and Kelvin force microscopy measurements. The results show how the band gap, the valence band maximum, the electron affinity and the work function all depend on the ND's size and nanoparticle surface properties. The present analysis, which combines information from both scanning tunneling spectroscopy and Kelvin force microscopy, should be applicable to any nanoparticle or surface that can be measured with scanning probe techniques.

Transfer doping of single isolated nanodiamonds, studied by scanning probe microscopy techniques

Science.gov (United States)

Bolker, Asaf; Saguy, Cecile; Kalish, Rafi

2014-09-01

The transfer doping of diamond surfaces has been applied in various novel two-dimensional electronic devices. Its extension to nanodiamonds (ND) is essential for ND-based applications in many fields. In particular, understanding the influence of the crystallite size on transfer doping is desirable. Here, we report the results of a detailed study of the electronic energetic band structure of single, isolated transfer-doped nanodiamonds with nanometric resolution using a combination of scanning tunneling spectroscopy and Kelvin force microscopy measurements. The results show how the band gap, the valence band maximum, the electron affinity and the work function all depend on the ND’s size and nanoparticle surface properties. The present analysis, which combines information from both scanning tunneling spectroscopy and Kelvin force microscopy, should be applicable to any nanoparticle or surface that can be measured with scanning probe techniques.

Transfer doping of single isolated nanodiamonds, studied by scanning probe microscopy techniques

International Nuclear Information System (INIS)

Bolker, Asaf; Kalish, Rafi; Saguy, Cecile

2014-01-01

The transfer doping of diamond surfaces has been applied in various novel two-dimensional electronic devices. Its extension to nanodiamonds (ND) is essential for ND-based applications in many fields. In particular, understanding the influence of the crystallite size on transfer doping is desirable. Here, we report the results of a detailed study of the electronic energetic band structure of single, isolated transfer-doped nanodiamonds with nanometric resolution using a combination of scanning tunneling spectroscopy and Kelvin force microscopy measurements. The results show how the band gap, the valence band maximum, the electron affinity and the work function all depend on the ND’s size and nanoparticle surface properties. The present analysis, which combines information from both scanning tunneling spectroscopy and Kelvin force microscopy, should be applicable to any nanoparticle or surface that can be measured with scanning probe techniques. (paper)

Vector model for polarized second-harmonic generation microscopy under high numerical aperture

International Nuclear Information System (INIS)

Wang, Xiang-Hui; Chang, Sheng-Jiang; Lin, Lie; Wang, Lin-Rui; Huo, Bing-Zhong; Hao, Shu-Jian

2010-01-01

Based on the vector diffraction theory and the generalized Jones matrix formalism, a vector model for polarized second-harmonic generation (SHG) microscopy is developed, which includes the roles of the axial component P z , the weight factor and the cross-effect between the lateral components. The numerical results show that as the relative magnitude of P z increases, the polarization response of the second-harmonic signal will vary from linear polarization to elliptical polarization and the polarization orientation of the second-harmonic signal is different from that under the paraxial approximation. In addition, it is interesting that the polarization response of the detected second-harmonic signal can change with the value of the collimator lens NA. Therefore, it is more advantageous to adopt the vector model to investigate the property of polarized SHG microscopy for a variety of cases

Multispectral digital lensless holographic microscopy: from femtosecond laser to white light LED

International Nuclear Information System (INIS)

Garcia-Sucerquia, J

2015-01-01

The use of femtosecond laser radiation and super bright white LED in digital lensless holographic microscopy is presented. For the ultrafast laser radiation two different configurations of operation of the microscope are presented and the dissimilar performance of each one analyzed. The microscope operating with a super bright white light LED in combination with optical filters shows very competitive performance as it is compared with more expensive optical sources. The broadband emission of both radiation sources allows the multispectral imaging of biological samples to obtain spectral responses and/or full color images of the microscopic specimens; sections of the head of a Drosophila melanogaster fly are imaged in this contribution. The simple, solid, compact, lightweight, and reliable architecture of digital lensless holographic microscopy operating with broadband light sources to image biological specimens exhibiting micrometer-sized details is evaluated in the present contribution. (paper)

Acoustic profilometry within polymers as performed by Brillouin microscopy

Energy Technology Data Exchange (ETDEWEB)

Sanctuary, R [Laboratoire Europeen de Recherche Universitaire Saarland-Lorraine (LERUSL) at the Centre Universitaire de Luxembourg, Departement des Sciences, Laboratoire 1.19, 162a avenue de la Faiencerie, L-1511 Luxembourg (Luxembourg); Bactavatchalou, Ravi [Universitaet des Saarlandes, Fakultaet fuer Physik und Elektrotechnik 7.2, Gebaeude 38, D-66041 Saarbruecken (Germany); Mueller, Ulrich [Universitaet des Saarlandes, Fakultaet fuer Physik und Elektrotechnik 7.2, Gebaeude 38, D-66041 Saarbruecken (Germany); Possart, W [Universitaet des Saarlandes, Fakultaet fuer Chemie, Pharmazie und Werkstoffwissenschaften 8.15, Gebaeude 22, D-66041 Saarbruecken (Germany); Alnot, P [Laboratoire de Physique des Milieux Ionises et Applications (LPMIA), UMR 7040-C.N.R.S, Universite de Nancy I, Bd des Aiguillettes, B.P. 239 F-54506 Vandoeuvre les Nancy (France); Krueger, J K [Universitaet des Saarlandes, Fakultaet fuer Physik und Elektrotechnik 7.2, Gebaeude 38, D-66041 Saarbruecken (Germany)

2003-11-07

Using high performance Brillouin spectroscopy we present a new technique, which enables us to perform acoustic microscopy with a spatial resolution of about 1 {mu}m. This technique, called Brillouin microscopy, is tested on several bulk- and film-like polymer samples.

Scanning Tunneling Microscopy - image interpretation

International Nuclear Information System (INIS)

Maca, F.

1998-01-01

The basic ideas of image interpretation in Scanning Tunneling Microscopy are presented using simple quantum-mechanical models and supplied with examples of successful application. The importance is stressed of a correct interpretation of this brilliant experimental surface technique

Direct observation for atomically flat and ordered vertical {111} side-surfaces on three-dimensionally figured Si(110) substrate using scanning tunneling microscopy