Confocal imaging analysis of ATP-induced Ca2+ response in individual endothelial cells of the artery in situ

1997 ◽  
Vol 272 (6) ◽  
pp. C1980-C1987 ◽  
Author(s):  
H. Ohata ◽  
Y. Ujike ◽  
K. Momose
Keyword(s):  
Endothelial Cells ◽  
Laser Scanning ◽  
Cultured Cells ◽  
Laser Scanning Confocal Microscopy ◽  
Confocal Imaging ◽  
Transient Increase ◽  
Similar Response ◽  
Imaging Analysis ◽  
Acetoxymethyl Ester

The mechanisms for mobilization of intracellular free Ca2+ have been studied in various types of isolated and cultured cells, but little is known about Ca2+ mobilization in individual cells in situ. We tried to establish imaging analysis of intracellular free Ca2+ concentration ([Ca2+]i) in individual cells loaded with the acetoxymethyl ester of fluo 3 in situ, using laser scanning confocal microscopy. The method permitted us to distinguish signals from endothelial and smooth muscle cells of guinea pig artery. Addition of ATP to the artery caused a transient increase in endothelial [Ca2+]i. It was concluded that the response was induced via P2Y purinoceptors, because adenosine 5'-O-(2-thiodiphosphate), but not UTP, caused a similar response independent of extracellular Ca2+. The percentage of cells that responded to ATP (1-10 microM) and the peak amplitude of the transient increase in [Ca2+]i were dose dependently increased. Using rapid xy-scanning and line-scanning modes, we confirmed that 10 microM ATP induced Ca2+ waves, at a rate of 10-30 microns/s, after a lag time of approximately 3 s. These results show that [Ca2+]i waves within endothelial cells are physiologically induced by ATP via P2Y purinoceptor, but not P2U purinoceptor, in aortic strips in situ. The method should be of use in the study of vascular physiology and pathophysiology.

In-situ Observation of Surface Relief Formation and Disappearance during Order-Disorder Transition of Equi-atomic CuAu alloy using Laser Scanning Confocal Microscopy

2004 ◽  
Vol 842 ◽  
Author(s):  
Seiji Miura ◽  
Hiroyuki Okuno ◽  
Kenji Ohkubo ◽  
Tetsuo Mohri
Keyword(s):  
Surface Relief ◽  
Laser Scanning ◽  
Laser Scanning Confocal Microscopy ◽  
Confocal Scanning Laser Microscopy ◽  
Strain Effect ◽  
In Situ Observation ◽  
Stress Effect ◽  
Scanning Confocal Microscopy ◽  
Crystallographic Orientation Relationship

ABSTRACTIn-situ observation of the formation and disappearance of the surface relief associated with the twinning during the order-disorder transitions among CuAu-I (L10), CuAu-II (PAP) and disordered fcc phases was conducted using Confocal Scanning Laser Microscopy equipped with a gold image furnace. The Retro effect was confirmed in poly-crystal samples, however no evidence was found in single-crystal samples. Also observed in poly-crystal samples are that the disordering temperature detected by the disappearing of relieves is different from grain to grain, and that grain boundary cracking alleviates the Retro effect. The observed phenomena were explained based on the crystallographic orientation relationship among grains investigated by FESEM/EBSD in terms of the elastic strain effect around grain boundaries induced by transition. It was confirmed that in each grain the surface relieves correspond to a set of two {011} planes having a <100> axis perpendicular to both planes in common. It was also found that the larger the average strain of two neighboring grains is, the lower the transition temperature. This observation was explained by the stress effect on the stability of a phase.

Three-Dimensional Laser-Scanning Confocal Microscopy of In Situ Hybridization in the Skin

1994 ◽  
Vol 16 (1) ◽  
pp. 44-51 ◽  
Author(s):  
Stephen E. Mahoney ◽  
Stephen W. Paddock ◽  
Louis C. Smith ◽  
Dorothy E. Lewis ◽  
Madeleine Duvic
Keyword(s):  
In Situ Hybridization ◽  
Confocal Microscopy ◽  
Laser Scanning ◽  
Three Dimensional ◽  
Laser Scanning Confocal Microscopy ◽  
Scanning Confocal Microscopy

Effect of Ca2+ agonists in the perfused liver: determination via laser scanning confocal microscopy

1999 ◽  
Vol 276 (2) ◽  
pp. R575-R585 ◽  
Author(s):  
Kentaro Motoyama ◽  
Irene E. Karl ◽  
M. Wayne Flye ◽  
Dale F. Osborne ◽  
Richard S. Hotchkiss
Keyword(s):  
Confocal Microscopy ◽  
Laser Scanning ◽  
Cyclopiazonic Acid ◽  
Isolated Hepatocytes ◽  
Laser Scanning Confocal Microscopy ◽  
Simultaneous Increase ◽  
Acetoxymethyl Ester ◽  
Scanning Confocal Microscopy ◽  
Or Groups ◽  
Periportal Area

Ca2+is a critical intracellular second messenger, but few studies have examined Ca2+ signaling in whole organs. The amplitude and frequency of Ca2+ oscillations encode important cellular information. Using laser scanning confocal microscopy in the indo 1 acetoxymethyl ester dye-loaded rat liver, we investigated the effect of various Ca2+ agonists that act at distinct mechanistic sites on Ca2+ signaling. Perfusion with suprathreshold doses of arginine vasopressin (AVP) (2–20 nM) caused a single Ca2+ wave that originated in the pericentral vein region and spread centrifugally to the periportal area. Lower doses of AVP (0.2–2 nM) caused multiple Ca2+ waves and Ca2+ oscillations. Perfusion with ATP (1.4–17.5 μM) caused rapid transient elevations in intracellular free Ca2+concentration ([Ca2+]i) occurring in isolated hepatocytes or groups of hepatocytes throughout the lobule and were of shorter duration than those due to AVP. Also in contrast to AVP, there was no specific anatomic location within the hepatic lobule that was more susceptible to ATP. Thapsigargin and cyclopiazonic acid did not cause a Ca2+ wave but rather produced a uniform and fairly simultaneous increase in [Ca2+]iin all hepatocytes in the lobule. Perfusion with 14 μM ryanodine produced a single transient spike in [Ca2+]iin a small number (<2%) of hepatocytes. Dantrolene, an inhibitor of Ca2+ release, reduced the increased [Ca2+]ioccurring after AVP. Insight into the mechanism of action of these Ca2+-active compounds on Ca2+ signaling in the intact liver is provided.

FRET-based voltage probes for confocal imaging: membrane potential oscillations throughout pancreatic islets

2005 ◽  
Vol 289 (1) ◽  
pp. C224-C229 ◽  
Author(s):  
Andrey Kuznetsov ◽  
Vytautas P. Bindokas ◽  
Jeremy D. Marks ◽  
Louis H. Philipson
Keyword(s):  
Membrane Potential ◽  
Laser Scanning ◽  
Islet Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Laser Scanning Confocal Microscopy ◽  
Confocal Imaging ◽  
Tight Coupling ◽  
Similar Time ◽  
Potential Oscillations

Insulin secretion is dependent on coordinated pancreatic islet physiology. In the present study, we found a way to overcome the limitations of cellular electrophysiology to optically determine cell membrane potential ( Vm) throughout an islet by using a fast voltage optical dye pair. Using laser scanning confocal microscopy (LSCM), we observed fluorescence (Förster) resonance energy transfer (FRET) with the fluorescent donor N-(6-chloro-7-hydroxycoumarin-3-carbonyl)-dimyristoylphosphatidyl-ethanolamine and the acceptor bis-(1,3-diethylthiobarbiturate) trimethine oxonol in the plasma membrane of essentially every cell within an islet. The FRET signal was approximately linear from Vm −70 to +50 mV with a 2.5-fold change in amplitude. We evaluated the responses of islet cells to glucose and tetraethylammonium. Essentially, every responding cell in a mouse islet displayed similar time-dependent changes in Vm. When Vm was measured simultaneously with intracellular Ca2+, all active cells showed tight coupling of Vm to islet cell Ca2+ changes. Our findings indicate that FRET-based, voltage-sensitive dyes used in conjunction with LSCM imaging could be extremely useful in studies of excitation-secretion coupling in intact islets of Langerhans.

Endothelial Cells Organize Fibrin Clots into Structures That Are More Resistant to Lysis

2005 ◽  
Vol 11 (3) ◽  
pp. 268-277 ◽  
Author(s):  
W. Gray Jerome ◽  
Stefan Handt ◽  
Roy R. Hantgan
Keyword(s):  
Endothelial Cells ◽  
Laser Scanning ◽  
Umbilical Vein ◽  
Survival Rates ◽  
The United States ◽  
Laser Scanning Confocal Microscopy ◽  
Scanning Confocal Microscopy ◽  
Endothelial Surface ◽  
Clot Structure ◽  
Fibrin Clots

Acute myocardial infarction is a major cause of death and disability in the United States. Introducing thrombolytic agents into the clot to dissolve occlusive coronary artery thrombi is one method of treatment. However, despite advances in our knowledge of thrombosis and thrombolysis, survival rates following thrombolytic therapy have not improved substantially. This failure highlights the need for further study of the factors mediating clot stabilization. Using laser scanning confocal microscopy of clots formed from fluorescein-labeled fibrinogen, we investigated what effect binding of fibrin to the endothelial surface has on clot structure and resistance to lysis. Fluorescent fibrin clots were produced over human umbilical vein endothelial cells (HUVEC) and the clot structure analyzed. In the presence of HUVEC, fibrin near the endothelial surface was more organized and occurred in tighter bundles compared to fibrin just 50 μm above. The HUVEC influence on fibrin architecture was blocked by inhibitory concentrations of antibodies to αVor β3integrin subunits. The regions of the clots associated with endothelial cells were more resistant to lysis than the more homogenous regions distal to endothelium. Thus, our data show that binding of fibrin to integrins on endothelial surfaces produces clots that are more resistant to lysis.

Interleukin-13 induces PSGL-1/P–selectin–dependent adhesion of eosinophils, but not neutrophils, to human umbilical vein endothelial cells under flow

Blood ◽  
2000 ◽  
Vol 95 (10) ◽  
pp. 3146-3152 ◽  
Author(s):  
Gerrit Woltmann ◽  
Claire A. McNulty ◽  
Grant Dewson ◽  
Fiona A. Symon ◽  
Andrew J. Wardlaw
Keyword(s):  
Endothelial Cells ◽  
Laser Scanning ◽  
Cultured Cells ◽  
Umbilical Vein ◽  
Allergic Inflammation ◽  
Similar Degree ◽  
Human Umbilical Vein ◽  
Laser Scanning Cytometry ◽  
Umbilical Vein Endothelial Cells ◽  
Necrosis Factor

Selective eosinophil accumulation is a hallmark of diseases such as asthma. In a model of chronic eosinophilic inflammation, we have previously shown that the tethering step in eosinophil adhesion is mediated by PSGL-1 binding to P-selectin. The Th2-associated cytokine IL-13 is of potential importance in allergic disease. We have therefore investigated whether IL-13 can mediate eosinophil binding to human umbilical vein endothelial cells (HUVEC) through P-selectin. IL-13 caused dose- and time-dependent increases of P-selectin expression, as assessed by flow and laser scanning cytometry. A similar degree of expression was observed with IL-4. There was no effect on E-selectin or ICAM-1 expression. Tumor necrosis factor- induced the expression of VCAM-1, E-selectin, and ICAM-1 but had no effect on P-selectin expression. IL-13 increased the production of mRNA for surface and soluble variants of P-selectin. Under flow conditions, eosinophils, but not neutrophils, showed enhanced binding to IL-13 and to IL-4–stimulated HUVEC compared to medium-cultured cells. Eosinophil adhesion was completely inhibited by a blocking monoclonal antibody against PSGL-1 and P-selectin. Anti–VLA-4 and anti–VCAM-1 antibodies inhibited binding to a lesser extent. Thus, at physiologic levels of expression induced by Th2 cytokines, P-selectin/PSGL-1 supported eosinophil but not neutrophil adhesion. This mechanism is likely to be a key event leading to the selective accumulation of eosinophils in allergic inflammation.

Photo-Induced Damages of Cytoplasmic and Mitochondrial Membranes by a [C60]Fullerene Malonic Acid Derivative

2007 ◽  
Vol 26 (3) ◽  
pp. 197-201 ◽  
Author(s):  
Xinlin Yang ◽  
Lin Chen ◽  
Xinge Qiao ◽  
Cuihong Fan
Keyword(s):  
Laser Scanning ◽  
Cytoplasmic Membrane ◽  
Biological Activities ◽  
Laser Scanning Confocal Microscopy ◽  
Laser Source ◽  
C60 Fullerene ◽  
Dependent Manner ◽  
Mitochondrial Membranes ◽  
Acetoxymethyl Ester ◽  
Fullerene Derivatives

The biological activities of fullerene derivatives have attracted much attention in the last decade. In this paper, effects of dimalonic acid C60 (DMA C60) on cytoplasmic membrane, intracellular calcium concentration ([Ca2+]i), and mitochondrial membrane in HeLa cells were studied by using laser scanning confocal microscopy together with fluorescent probes propidium iodide (PI), fluo-3 acetoxymethyl ester (fluo-3 AM), and tetramethyl rhodamine methyl ester (TMRM). The data showed that under laser irradiation produced by a Kr/Ar laser source with a low power less than 1 mW, DMA C60 might induce damages against both cytoplasmic and mitochondrial membranes in a time- and dose-dependent manner. Prior to leakage of cytoplasmic membrane, a transient increase in [Ca2+]i occurred due to influx of calcium from the culture medium. These data provided some novel clues to explain the mechanisms involved in the photo-induced cytotoxicity of fullerene derivatives.

In Situ Observation of Semisolid Fe-2.5C-1.5Si Gray Cast Iron

2016 ◽  
Vol 256 ◽  
pp. 63-68
Author(s):  
Davi Munhoz Benati ◽  
Kazuhiro Ito ◽  
Kazuyuki Kohama ◽  
Hajime Yamamoto ◽  
Eugênio José Zoqui
Keyword(s):  
Cast Iron ◽  
Liquid Phase ◽  
Laser Scanning ◽  
Gray Cast Iron ◽  
Solid Phase ◽  
Laser Scanning Confocal Microscopy ◽  
Raw Material ◽  
Gray Cast ◽  
Heating Process

Fe-2.5C-1.5Si gray cast iron evaluated in previous works exhibited promising potential as semisolid raw material presenting low levels of maximum stress and viscosity, similar to Al-Si alloys. This work is intended to investigate phase transformations and liquid phase formation for the Fe-2.5C-1.5Si gray cast iron in order to understand the performance of the alloy during the semisolid processing. Thus in situ heating experiments via high temperature laser scanning confocal microscopy were performed to analyze the solid-to-liquid transition. At room temperature alloy presented a matrix of pearlite and ferrite with type D flake graphite. During the heating process the main transformations observed were graphite precipitation on the austenite grain boundaries, graphite precipitates and flakes graphite growing and coarsening with the increasing of temperature and the beginning of melt around 1140°C. Coarsened flakes at high temperatures resulted in a liquid continuous network after melting, thereby the liquid phase was formed surrounding and wetting homogeneously the solid phase. This favors the detachment of grains from each other and leads to the intended solid globules immersed in liquid.

Analysis of the 355 Aluminium Alloy Microstructure for Application in Thixoforming

2019 ◽  
Vol 285 ◽  
pp. 277-282
Author(s):  
Leandro Cássio de Paula ◽  
Shun Tokita ◽  
Kota Kadoi ◽  
Hiroshige Inoue ◽  
Eugênio José Zoqui
Keyword(s):  
Aluminium Alloy ◽  
Laser Scanning ◽  
Ostwald Ripening ◽  
Phase Particle ◽  
Thermodynamic Characteristics ◽  
Laser Scanning Confocal Microscopy ◽  
Primary Phase ◽  
Raw Material ◽  
In Situ Observation

The 355 aluminium alloy is known to have excellent thermodynamic characteristics that render it suitable as a raw material for rheocasting and thixoforming. However, besides the controllable transition from solid to liquid phase, the refined microstructure required in the semisolid range is one of the key factors with a strong influence on the rheology of the material. This paper intends to analyse the in situ behaviour of the microstructure, in terms of morphological change, using high-temperature laser scanning confocal microscopy. The 355 alloy was prepared via conventional casting, and refined with a 30-s exposition via ultrasonic melt treatment (UST - 20 Hz, 2 kW). The material was reheated up to the thixoforming target temperature of 595 °C at which it was maintained for 0, 30, 60, 90, and 120 s, after which all the samples were cooled in water. The samples subjected to UST prior to the heat treatment were more refined in terms of microstructural evolution; they exhibited reduction in grain size (~107 ± 16 μm), smallest primary phase particle size (~81 ± 7 μm), and high circularity shape factor (~0.59 ± 0.19 μm). In situ observation methods were employed to analyse evolution mechanisms such as Ostwald ripening and coalescence.

scholarly journals An In Situ Hybridization Histochemistry Technique Allowing Simultaneous Visualization by the Use of Confocal Microscopy of Three Cellular mRNA Species in Individual Neurons

1998 ◽  
Vol 46 (6) ◽  
pp. 753-759 ◽  
Author(s):  
Michel Grino ◽  
Alfredo J. Zamora
Keyword(s):  
Confocal Microscopy ◽  
Laser Scanning ◽  
Simultaneous Detection ◽  
Laser Scanning Confocal Microscopy ◽  
Mrna Species ◽  
Scanning Confocal Microscopy ◽  
Detection Systems ◽  
Low Concentrations ◽  
Simultaneous Visualization

We present a specific and sensitive method for simultaneous detection of three mRNA species in individual neurons. The method relies on the use of riboprobes labeled with [35S]-UTP, digoxigenin-UTP, or biotin-UTP. The nonradioactive probes were sequentially revealed by incubation with anti-digoxigenin immunoglobulins or streptavidin conjugated to peroxidase, followed by the use of fluorochrome-labeled tyramides as peroxidase substrates. The radioactive probe was revealed by conventional autoradiography. There was no interaction among the different probes or the various detection systems. We demonstrate the use of this method by illustrating on laser scanning confocal microscopy the co-localization of the mRNAs coding for corticotropin-releasing factor (CRF), arginine vasopressin (AVP), or peptidylglycine α-amidating monooxygenase (PAM) in rat hypothalamic paraventricular nucleus (PVN) and its modulation by endogenous glucocorticoids. Our results suggest that this method could be used not only to study the regulation of the hypothalamo-pituitary-adrenal axis but also in various models in which mRNAs are present at low concentrations.

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