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At least 847 records · Page 47Linked to original sources
PubMed2002

MRI and fluorescence microscopy of the acute vascular response to VEGF165: vasodilation, hyper-permeability and lymphatic uptake, followed by rapid inactivation of the growth factor.

Vascular endothelial growth factor (VEGF) is one of the key growth factors regulating tumor angiogenesis and thus it is one of the primary targets for antiangiogenic therapy. The long-term effects of VEGF include induction of proliferation and migration of endothelial cells, tube formation and maintenance of the immature capillaries. The early effects of VEGF include vasodilation and increased permeability. We hypothesize that the early responses to VEGF can serve to develop a quantitative measure of the activity of VEGF, and therefore may be applicable for monitoring the efficacy of systemic suppression of VEGF signaling during antiangiogenic therapy. For that end we tested the ability of MRI and fluorescence microscopy to detect the early response to intradermal VEGF165 in nude mice. VEGF-induced local vasodilation and increased permeability was detected by intravenous administration of macromolecular biotin-BSA-GdDTPA(23) 30 min after intradermal administration of VEGF. Contrast leak showed saturation kinetics. Delayed contrast administration (90 min after intradermal administration of VEGF) resulted in low contrast leak and demonstrated that the saturation kinetics is not due to contrast equilibration between plasma and the interstitial space, but rather is due to suppression of vascular permeability. Permeability was restored by a second bolus of VEGF, showing that the saturation kinetics is primarily due to inactivation of the growth factor. Confocal microscopy of fluorescent BSA-FITC confirmed the permeability changes monitored by MRI. Moreover, confocal microscopy showed efficient lymphatic uptake of the extravasated contrast material specifically in regions of VEGF induced hyper-permeability.

Animals↗
PubMed2006

Major signal increase in fluorescence microscopy through dark-state relaxation.

We report a substantial signal gain in fluorescence microscopy by ensuring that transient molecular dark states with lifetimes >1 micros, such as the triplet state relax between two molecular absorption events. For GFP and Rhodamine dye Atto532, we observed a 5-25-fold increase in total fluorescence yield before molecular bleaching when strong continuous-wave or high-repetition-rate pulsed illumination was replaced with pulses featuring temporal pulse separation >1 micros. The signal gain was observed both for one- and two-photon excitation. Obeying dark or triplet state relaxation in the illumination process signifies a major step toward imaging with low photobleaching and strong fluorescence fluxes.

Green Fluorescent Proteins↗
PubMed1999

The UVA light used during the fluorescence microscopy assay affects the level of intracellular calcium being measured in experiments with electric-field exposure.

In the present paper, the induction of calcium signals in neuroblastoma cells, cells of T-cell leukemia, and osteogenic sarcoma cells were investigated in relation to the UVA irradiation used in fluorescence microscopy. Methods were developed to measure both the mean UVA irradiance and the intensity profile in the UVA-illuminated area of the microscope. This allowed us to calculate the applied UVA radiant exposure of the cells during each experiment. This investigation was undertaken because of the conflicting results in the literature on the effects of electromagnetic fields on the signals of the calcium-sensitive fluorescence probe FURA-2 in lymphocytes. Taking into account that each group used a different system with different optics and lamps, these conflicting results are now at least partially understandable. Our measurements indicate that in a typical experiment with FURA-2 the cells were irradiated with up to 776 kJ m(-2) during 25 min of exposure to UVA light. This causes changes in intracellular free Ca(2+) concentrations ([Ca(2+)](i)). Designating cells in which the [Ca(2+)](i) was distinctly increased during the experiment as "responding", we found Hill-type dependences on the irradiance. Jurkat cells showed a 50% response even at 10 kJ m(-2) and osteosarcoma cells at about 60 kJ m(-2), whereas neuroblastoma cells even at the maximum possible dose responded only minimally. In the case of neuroblastoma cells, we found a dependence of this effect on the CO(2) partial pressure during the preincubation. An electrical treatment with an a.c. field (5 kHz sinusoidal, amplitude modulation 16 Hz 100%, 800 V m(-1), 5 min) had a significant effect on intracellular calcium in neuroblastoma cells only in the case of cells that were not pretreated with CO(2) with high fluences of UVA irradiation. In conclusion, these results indicate that the possibility of UVA artifacts must be considered in all experiments using fluorescence microscopy. Furthermore, our results lead to the hypothesis that oxidative stress could be the link between UVA and electric-field effects.

Animals↗
PubMed1994

Detection of cholesterol associated with calcium mineral using confocal fluorescence microscopy.

BACKGROUND: Cholesterol and calcium are prominant components within human atherosclerotic lesions. Both accumulate predominantly within the central core region of lesions. Because of similarities in some crystallographic faces of cholesterol monohydrate and calcium apatite, it has been previously proposed that deposition of one may nucleate the deposition of the other. EXPERIMENTAL DESIGN: In this study, we have used the technique of confocal fluorescence microscopy to assess the spatial orientation of cholesterol in association with calcium mineral. Localization of cholesterol within mineral was carried out by staining cholesterol with the fluorescent probe, filipin. RESULTS: With this technique, it was possible to localize cholesterol associated with the surface of hydroxyapatite seeds, cholesterol incorporated within calcium phosphate-cholesterol agglomerates produced in vitro, and cholesterol within apatite isolated from human atherosclerotic lesions. CONCLUSIONS: The presence of cholesterol within the center of calcified granules from atherosclerotic plaque suggests that cholesterol or associated lipids may act to nucleate the deposition of apatite. Confocal fluorescence microscopy should be a useful technique by which to study the relationship of cholesterol associated with calcium minerals that occur not only in atherosclerotic blood vessels, but also in gallstones, and calcified cardiac valves.

Apatites↗
PubMedDate not supplied

Characterization of novel histidine-tagged Tat-peptide complexes dual-labeled with (99m)Tc-tricarbonyl and fluorescein for scintigraphy and fluorescence microscopy.

To enable concurrent whole body scintigraphy and direct imaging of subcellular localization of permeation peptides, dual-labeled Tat-peptides useful for both radiometric analysis and fluorescence microscopy are desired for molecular imaging applications. Thus, novel dual-labeled D-Tat-peptides comprising Tat-basic domain (hgrkkrrqrrrgc), C-terminus conjugated with fluorescein-5-maleimide (FM) and N-terminus chelated with [(99m)Tc(CO)(3)] via histidine coordination, were synthesized and characterized. In human Jurkat cells, radiotracer uptake and washout studies revealed concentration-dependent accumulation of the dual-labeled Tat-peptide within cells. Subcellular localization of Tat-peptide was confirmed by fluorescence microscopy using an analogous [Re(CO)(3)] dual-labeled Tat-peptide. As seen with C-terminus single-labeled Tat-peptides, localization to the nucleoli was observed with the dual-labeled Tat-peptide, suggesting that the mechanism of Tat-peptide uptake and localization was not dependent on free peptide termini at either end. In Balb/c mice, biodistribution studies performed with the dual-labeled Tat-peptide showed fluorescence intensity by microscopic analysis that visually confirmed and correlated directly with scintigraphic and radiometric data. Of note, following intravenous administration, little brain penetration of these permeation sequences was observed in vivo. His[(99m)Tc(CO)(3)]-, DTPA[(99m)Tc(CO)(3)]-, and epsilon-lys-gly-cys[(99m)Tc(O)]-labeled Tat-peptides showed significant pharmacokinetic differences in liver and kidney depending on labeling strategy, indicating that Tat-peptide biodistribution can be impacted by the chelation moiety coordinated with (99m)Tc. Thus, we have shown that dual-labeled (99m)Tc-tricarbonyl Tat-peptide-FM conjugates can be conveniently synthesized and enable direct comparison of quantitative radiometric and qualitative fluorescence data both in vitro as well as in vivo.

Animals↗
PubMed1994

In vivo fluorescence microscopy for the assessment of microvascular reperfusion injury in small bowel transplants in rats.

With the use of in vivo fluorescence microscopy we have analyzed microvascular reperfusion injury of small bowel isograft transplants in rats. Following 1 hr cold storage in University of Wisconsin solution, the small bowel was transplanted heterotopically, and the intestinal microcirculation was quantitatively analyzed 20-60 min after onset of reperfusion. The intestinal grafts' capillary perfusion of both the mucosa and the circular and longitudinal muscles was not found altered when compared with the intestinal capillary perfusion of sham-operated controls. In contrast, leukocyte-endothelial cell interaction, including leukocyte rolling (40 +/- 5%) and sticking (280 +/- 100 mm-2) in submucosal postcapillary venules, was significantly increased when compared with nontransplanted controls (12 +/- 8% and 20 +/- 10 mm-2, P < 0.01 and P < 0.05, respectively). Leukocyte-endothelial cell interaction was associated with a marked alteration of lymphatic capillary drainage, as indicated by the low functional density of lymphatic microvessels of 10.2 +/- 6.1 cm-1 (P < 0.01 vs. sham-operated controls (39.2 +/- 6.1 cm-1)). From these results we propose that leukocyte-endothelial cell interaction, not capillary "no-reflow," is the primary step in the manifestation of microvascular reperfusion injury following a short period of cold ischemia in small bowel grafts.

Animals↗
PubMed1993

In vivo fluorescence microscopy of microcirculation in the renal cortex of mice. Part II. Effects of mannitol and contrast media infusions.

In vivo fluorescence microscopy was used for experimental studies of renal cortical microcirculation in 46 mice. The cortical circulation was studied after i.v. infusions of mannitol of various osmolalities as well as nonionic low osmolar (iohexol) and ionic high osmolar (metrizoate) contrast media. All infusions produced an increase in the number of capillaries with increased blood flow, significantly more pronounced after the infusion of iohexol, metrizoate and mannitol 1.46 mol/l than after the infusion of mannitol 0.3 mol/l. However, the renal cortical blood flow was inhomogeneous with respect to different capillaries. While the blood flow was increased in some capillaries it was to some extent simultaneously decreased in others, significantly more after infusion of metrizoate than after infusion of mannitol 0.3 mol/l.

Animals↗
PubMed1991

Macrophage phagocytosis: use of fluorescence microscopy to distinguish between extracellular and intracellular bacteria.

One of the challenges of phagocytosis research is to differentiate bacteria adherent to a host cell from bacteria which the cell has internalized. To address this question, various techniques such as fluorescence microscopy, electron microscopy, and flow cytometry have been used. We have adapted a flow cytometric method (Fattorossi et al., 1989) to use fluorescence microscopy for studying phagocytosis of fluorescein-labeled Listeria by inflammatory mouse peritoneal macrophages. In this assay, ethidium bromide is used as a quenching agent and is added to cells after they have phagocytosed labeled bacteria. Ethidium bromide causes extracellular FITC-labeled Listeria to fluoresce red-orange, whereas intracellular bacteria are not exposed to the dye and remain green. This process allows distinction between intracellular and extracellular bacteria by simultaneous visualization of both populations.

Animals↗
PubMed1987

An inexpensive dual-excitation apparatus for fluorescence microscopy.

Ion sensitive indicator molecules can be employed in conjunction with fluorescence microscopy in single cells to measure rapid changes in the intracellular concentration of several ionic species. A number of these probes (e.g. fura-2) require the capability of measuring emission intensity at two excitation wavelengths to quantitate properly intracellular ion concentration. We have developed a simple dual-excitation apparatus for use in such applications. The apparatus switches the excitation filter within 150 ms. This economical apparatus is well suited in situations where the ionic concentration of interest is changing relatively slowly. Moreover, by synchronizing the device's action with an external stimulus, rapid and reproducible ionic changes in excitable tissue also can be measured.

Animals↗
PubMed2005

Intravital imaging of tumour vascular networks using multi-photon fluorescence microscopy.

The blood supply of solid tumours affects the outcome of treatment via its influence on the microenvironment of tumour cells and drug delivery. In addition, tumour blood vessels are an important target for cancer therapy. Intravital microscopy of tumours growing in 'window chambers' in animal models provides a means of directly investigating tumour angiogenesis and vascular response to treatment, in terms of both the morphology of blood vessel networks and the function of individual vessels. These techniques allow repeated measurements of the same tumour. Recently, multi-photon fluorescence microscopy techniques have been applied to these model systems to obtain 3D images of the tumour vasculature, whilst simultaneously avoiding some of the problems associated with the use of conventional fluorescence microscopy in living tissues. Here, we review the current status of this work and provide some examples of its use for studying the dynamics of tumour angiogenesis and vascular function.

Animals↗
PubMed2001

Highly nonlinear photodamage in two-photon fluorescence microscopy.

Two-photon fluorescence excitation is being increasingly used in laser scan microscopy due to very low photodamage induced by this technique under normal operation. However, excitation intensity has to be kept low, because nonlinear photodamage sets in when laser power is increased above a certain threshold. We studied this kind of damage in bovine adrenal chromaffin cells, using two different indicators of damage: changes in resting [Ca(2+)] level and the degranulation reaction. In agreement with previous studies, we found that, for both criteria, damage is proportional to the integral (over space and time) of light intensity raised to a power approximately 2.5. Thus, widening the laser pulse shape at constant average intensity both in time and in focal volume is beneficial for avoiding this kind of damage. Both measures, of course, reduce the two-photon fluorescence excitation. However, loss of signal can be compensated by increasing excitation power, such that, at constant damaging potential, signals may be even larger with long pulses and large focal volumes, because the exponent of the power law of damage is higher (mu approximately 2.5) than that of the two-photon signal (mu approximately 2).

Adrenal Glands↗
PubMed1975

Ultrastructural and fluorescence microscopy study of a case of acute erythremia.

The authors studies in fluorescence microscopy and electronic microscopy the dysmorphic erythroblasts of a case of acute erythremia. They found evidence of granular fluorescence on the smears, corresponding to the presence of prophyrins and proteinaceous cristallin structures on the ultrathin sections. It seems to be possible to establish a morphologic correlation between the fluorescent granules and the cristallin structures. Thus, the hypothesis of poryphrins adsorption on cristallin structures may be proposed.

Acute Disease↗
PubMed1991

Fluorescence microscopy of phospholipid monolayer phase transitions.

Over many years, a detailed picture of the phase transitions in phospholipid monolayers at the air-water interface has been constructed from extensive studies of the force-area, viscoelastic and surface potential properties of phospholipid monolayers, yet the microscopic nature of the transitions has remained obscure. Recent investigations have focused specifically on these aspects. Through the use of fluorescence microscopy, electron diffraction and X-ray scattering experiments, in combination with data obtained by classical methods, a wealth of new information regarding the properties of monolayers undergoing phase transitions has been generated. Direct observation of fluid-solid phase coexistence at the air-water interface has been achieved with fluorescence microscopy and on solid supports with electron microscopy. The fluid-solid coexistence region has been studied most thoroughly to date, but regions of gas-fluid and fluid-fluid phase coexistence have also been detected. Numerous factors govern the properties of the coexistence region: however, the prominent features can be explained in terms of a competition between forces: long-range electrostatic forces and short-range attractive forces. In this review these recent experimental findings and theoretical interpretations are summarized.

Electrochemistry↗
PubMed1989

Fluorescence microscopy in three dimensions.

The combination of the specificity provided by fluorescence microscopy and the ability to quantitatively analyze specimens in three dimensions allows the fundamental organization of cells to be probed as never before. Key features in this emergent technology have been the development of a wide variety of fluorescent dyes or fluorescently labeled probes to provide the requisite specificity. High-quality, cooled charge-coupled devices have recently become available. Functioning as nearly ideal imagers or "electronic film," they are more sensitive than photomultipliers and provide extraordinarily accurate direct digital readout from the microscope. Not only is this precision crucial for accurate quantitative imaging such as that required for the ratioing necessary to determine intracellular ion concentrations, but it also opens the way for sophisticated image processing. It is important to realize that image processing isn't simply a means to improve image aesthetics, but can directly provide new, biologically important information. The impact of modern video microscopy techniques (Allen, 1985; Inoué, 1986) attests to the fact that many biologically relevant phenomena take place at the limits of conventional microscopy. Image processing can be used to substantially enhance the resolution and contrast obtainable in two dimensions, enabling the invisible to be seen and quantitated. Cells are intrinsically three-dimensional. This can simply be a nuisance because of limited depth of focus of the microscope or it could be a fundamental aspect of the problem being studied. In either case, image processing techniques can be used to rapidly provide the desired representation of the data. In this chapter we have discussed the nature of image formation in three dimensions and dealt with several means to remove contaminating out-of-focus information. The most straightforward of these methods uses only information from adjacent focal planes to correct the central one. This approach can be readily applied to virtually any problem and with most commonly available image processing hardware to provide a substantially deblurred image in almost real time. In addition to covering more sophisticated algorithms where the utmost in three-dimensional imaging is required, we have developed a method for extremely rapidly and accurately producing an in-focus, high-resolution "synthetic projection" image from a thick specimen. This is equivalent to that produced by a microscope having the impossible combination of a high-NA objective lens and an infinite depth of focus. A variation on this method allows efficient calculation of stereo pairs.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1999

Automation of functional assays by flow injection fluorescence microscopy.

Bead-injection spectroscopy is a novel technique that uses immobilized eukaryotic cells on microbeads as a renewable biosensor for fluorescence microscopy. The use of a flow injection instrument allows fast functional assays that generate full kinetic characterization of a drug. Because the cell population is automatically replaced for each assay, variability is minimized, thus allowing greater accuracy.

Biotechnology↗
PubMed2004

Ionic effect on combing of single DNA molecules and observation of their force-induced melting by fluorescence microscopy.

Molecular combing is a powerful and simple method for aligning DNA molecules onto a surface. Using this technique combined with fluorescence microscopy, we observed that the length of lambda-DNA molecules was extended to about 1.6 times their contour length (unextended length, 16.2 microm) by the combing method on hydrophobic polymethylmetacrylate coated surfaces. The effects of sodium and magnesium ions and pH of the DNA solution were investigated. Interestingly, we observed force-induced melting of single DNA molecules.

Bacteriophage lambda↗