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At least 955 records · Page 53Linked to original sources
PubMed2005

Effect of isoproterenol on the regulation of rabbit airway ciliary beat frequency measured with high-speed digital and fluorescence microscopy.

To investigate how isoproterenol, a beta-adrenergic agonist, regulates airway ciliary beat frequency (CBF), we simultaneously quantified changes in rabbit airway CBF and intracellular calcium concentration ([Ca2+]i) in response to isoproterenol (100 micromol/L) by using high-speed (240 frames per second) and fluorescence microscopy. At approximately 30 degrees C, isoproterenol induced a change in CBF that could be separated into two parts. First, isoproterenol induced a moderate increase in the basal CBF that was calcium-independent. This response was unaffected by buffering the [Ca2+]i with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrapotassium salt (BAPTA). Second, isoproterenol induced a transient increase in CBF that was superimposed on the increased basal CBF and correlated with a simultaneous transient increase in [Ca2+]i. This transient increase in CBF was abolished by BAPTA. We conclude that isoproterenol initially increases CBF through a calcium-independent mechanism, probably via protein kinase A, and subsequently through a calcium-dependent mechanism mediated by an increase in [Ca2+]i.

Adrenergic beta-Agonists↗
PubMed1980

Examination of human semen infected with Ureaplasma urealyticum by fluorescence microscopy.

Semen specimens from men complaining of involuntary infertility were cultured for Ureaplasma urealyticum and large-colony mycoplasmas. Samples positive for ureaplasmas (but negative for large-colony mycoplasmas) were washed and stained with a DNA-binding fluorochrome (Hoechst No. 33258) and examined by fluorescence microscopy. DNA-containing particles of a size consistent with ureaplasmas were found adherent to the heads and tails of spermatozoa as well as to exfoliated urethral epithelial cells when present. The number of spermatozoa with adherent particles varied widely in different clinical samples, ranging from less than 1% to 20%. In a preliminary study of the possible use of this technique as an aid in diagnosing genital ureaplasma infection, we were able to correctly identify 13 of 15 positive samples prior to culture.

Animals↗
PubMed2001

Investigation of microcontact transfer of proteins from a selectively plasma treated elastomer stamp by fluorescence microscopy and force microscopy.

Selective plasma treatment of the recessed regions of the elastomer stamps is shown to alter the resultant protein patterns. Fluorescence microscopy is demonstrated to be an excellent tool to discriminate between regions of microcontact printed fluorescent dye-labelled albumin in polystyrene. Atomic force microscopy and shear force microscopy are used to provide high-resolution images of the patterned protein layers. The formation and characteristics of the patterns formed by these alternative strategies is discussed.

Animals↗
PubMed1993

Analysis of the interleukin 6 receptor on normal human keratinocytes by digital imaging fluorescence microscopy.

The interleukin 6 receptor (IL 6 receptor) on normal human keratinocytes was analyzed by digital imaging fluorescence microscopy (DIFM). Non-fixed keratinocytes were incubated with monoclonal anti-IL 6 receptor antibody (MT18). The fluorescence intensity was quantitatively determined by an interactive laser cytometer (ACAS 470). IL 6 receptor-positive cells showed high density staining with the specific antibody in the center of the elevated cell surface. Therefore, DIFM appears to be an effective method for study of the distribution of IL 6 receptors at the single living cell level.

Cells, Cultured↗
PubMed1988

Carboxyfluorescein transfer across the blood-retinal barrier evaluated by quantitative fluorescence microscopy: comparison with fluorescein.

Carboxyfluorescein levels in ocular tissues of normal rats were measured using quantitative fluorescence microscopy and compared with fluorescein levels to determine the extent to which blood-retinal barrier permeability is affected by the difference in lipid solubility of these two dyes. Retinal fluorescence intensity measurements at 2 min after i.v. dye injection were very much lower for carboxyfluorescein than for fluorescein despite similar plasma free dye concentrations. Marked leakage of dye from the optic disc into peripapillary retina was identified. At 1- and 2 hr, retinal levels of the two dyes became more similar, because fluorescein was removed from retina faster than carboxyfluorescein. After sodium-iodate-induced damage of the pigment epithelium, high levels of both dyes were evident in retina, but carboxyfluorescein was localized chiefly within extracellular space whereas fluorescein also densely stained cell somata. The fluorescence intensity levels recorded, which are proportional to the total mass of dye in the tissue, were correspondingly lower for carboxyfluorescein than for fluorescein, indicating that they were markedly affected by the different distribution of the two dyes. To relate tissue fluorescence intensity directly to dye concentration in the extracellular fluid, measurements were obtained from isolated retinas incubated in dye solutions of known concentration. Log-log plots demonstrated a linear relation between fluorescence intensity and medium concentration for both dyes, but retinal fluorescence of carboxyfluorescein, in correspondence with its limited distribution in the tissue space, was consistently less than that of fluorescein. The ratio of carboxyfluorescein to fluorescein fluorescence varied with the retinal layers but was constant for each layer over the concentration range tested. These fluorescence intensity ratios then were used to adjust the in vivo data so that comparison between the two dyes more closely reflected their extracellular dye concentration. With this correction the amount of carboxyfluorescein present in outer retina shortly after dye injection was approx. 1/10 that of fluorescein, indicating that carboxyfluorescein penetrates the pigment epithelium less readily than fluorescein, as expected from the difference in lipid solubility of the two dyes. However, fluorescence of both dyes in retina and presumably in vitreous humor eventually reached similar levels. This is attributed to entry of the dyes at sites of barrier discontinuity, as at the optic disc, and by a difference in their rates of removal from the intraocular compartment.

Animals↗
PubMed1999

An evaluation of two-photon excitation versus confocal and digital deconvolution fluorescence microscopy imaging in Xenopus morphogenesis.

The ability to visualize cell motility occurring deep in the context of opaque tissues will allow many currently intractable issues in developmental biology and organogenesis to be addressed. In this study, we compare two-photon excitation with laser scanning confocal and conventional digital deconvolution fluorescence microscopy, using the same optical configuration, for their ability to resolve cell shape deep in Xenopus gastrula and neurula tissues. The two-photon microscope offers better depth penetration and less autofluorescence compared to confocal and conventional deconvolution imaging. Both two-photon excitation and confocal microscopy also provide improved rejection of "out-of-focus" noise and better lateral and axial resolution than conventional digital deconvolution microscopy. Deep Xenopus cells are best resolved by applying the digital deconvolution method on the two-photon images. We have also found that the two-photon has better depth penetration without any degradation in the image quality of interior sections compared to the other two techniques. Also, we have demonstrated that the quality of the image changes at different depths for various excitation powers.

Animals↗
PubMed2004

Comparison of fluorescence microscopy and solid-phase cytometry methods for counting bacteria in water.

Total direct counts of bacterial abundance are central in assessing the biomass and bacteriological quality of water in ecological and industrial applications. Several factors have been identified that contribute to the variability in bacterial abundance counts when using fluorescent microscopy, the most significant of which is retaining an adequate number of cells per filter to ensure an acceptable level of statistical confidence in the resulting data. Previous studies that have assessed the components of total-direct-count methods that contribute to this variance have attempted to maintain a bacterial cell abundance value per filter of approximately 10(6) cells filter(-1). In this study we have established the lower limit for the number of bacterial cells per filter at which the statistical reliability of the abundance estimate is no longer acceptable. Our results indicate that when the numbers of bacterial cells per filter were progressively reduced below 10(5), the microscopic methods increasingly overestimated the true bacterial abundance (range, 15.0 to 99.3%). The solid-phase cytometer only slightly overestimated the true bacterial abundances and was more consistent over the same range of bacterial abundances per filter (range, 8.9 to 12.5%). The solid-phase cytometer method for conducting total direct counts of bacteria was less biased and performed significantly better than any of the microscope methods. It was also found that microscopic count data from counting 5 fields on three separate filters were statistically equivalent to data from counting 20 fields on a single filter.

Biomass↗
PubMed1998

pHi and pHo at different depths in perfused myocardium measured by confocal fluorescence microscopy.

Confocal microscopy and the H+-sensitive fluorophore carboxyseminaphthorhodafluor-1 (SNARF-1) were used to measure either intracellular pH (pHi) or extracellular pH (pHo) in isolated, arterially perfused rabbit papillary muscles. Single-excitation, dual-emission fluorescent images of the endocardial surface and underlying myocardium to a depth of 300 micron were simultaneously recorded from perfused cylindrical muscles suspended in a controlled atmosphere oriented oblique to the focal plane. Contraction was inhibited by the addition of butanedione monoxime. In separate muscles, pHo was measured during continuous perfusion of SNARF-1 free acid. pHi measurements were made after the muscle was loaded with SNARF-1/AM and the extracellular space was cleared of residual fluorophore. Initial experiments demonstrated the uniformity of ratiometric measurements as a function of pH, image depth, and fluorophore concentration, thereby establishing the potential feasibility of this method for quantitative intramural pH measurements. In subsequent experiments, the method was validated in isolated, arterially perfused rabbit papillary muscle during normal arterial perfusion and as pHi and pHo were altered by applying CO2 externally, exchanging HEPES and bicarbonate buffers, and changing pHi with NH4Cl washout. We conclude that in situ confocal fluorescent microscopy can measure pHi and pHo changes at the endocardial surface and deeper endocardial layers in arterially perfused ventricular myocardium. This method has the potential to study pHi regulation in perfused myocardium at boundaries where diffusion of gases, metabolites, and peptides are expected to modify processes that regulate pHi.

Acidosis↗
PubMed1990

Two-photon laser scanning fluorescence microscopy.

Molecular excitation by the simultaneous absorption of two photons provides intrinsic three-dimensional resolution in laser scanning fluorescence microscopy. The excitation of fluorophores having single-photon absorption in the ultraviolet with a stream of strongly focused subpicosecond pulses of red laser light has made possible fluorescence images of living cells and other microscopic objects. The fluorescence emission increased quadratically with the excitation intensity so that fluorescence and photo-bleaching were confined to the vicinity of the focal plane as expected for cooperative two-photon excitation. This technique also provides unprecedented capabilities for three-dimensional, spatially resolved photochemistry, particularly photolytic release of caged effector molecules.

Animals↗
PubMed2000

New insights in the cellular processing of platinum antitumor compounds, using fluorophore-labeled platinum complexes and digital fluorescence microscopy.

The cellular distribution and processing pathways of two platinum compounds, modeling the antitumor drug cisplatin (cDDP) in human osteosarcoma (U2-OS) cells is reported. A [Pt(en)Cl] entity has been covalently linked to a carboxyfluorescein diacetate (CFDA) moiety and to a dinitrophenyl (DNP) moiety. The two different constructs were administered to living cell cultures that were analyzed using digital fluorescence microscopy. The non-fluorescent CFDA construct becomes fluorescent after cellular uptake and subsequent acetate hydrolysis by esterases, and is therefore suitable to monitor platinum in living cells; the DNP construct can be visualized by immunocytochemistry and consequently serves as a control. Both complexes were readily internalized by the cells, and localized throughout the whole cell. After 2-3 h the complex accumulated in the nucleus, but 6-8 h after incubation a punctuate staining of a cytoplasmic region was observed, that persisted and became more pronounced after 24 h. The overall fluorescence in the cell decreased over time, implying a secretion of the platinum complex. Surprisingly, the accumulation remained visible after 72 h. Co-localization experiments with a Golgi apparatus-selective stain indicate the involvement of Golgi vesicles in intracellular processing of cisplatin-derived complexes. Immunocytochemical studies, using the DNP derivative, resulted in very similar images as obtained with the CFDA construct. CFDA-boc (a non-platinum-containing fluorescein derivative) was used as control: a faint staining throughout the whole cell was observed. Cisplatin-resistant U2-OS/Pt cells showed staining patterns very similar to the U2-OS cells using both platinum constructs. This study illustrates that only a very small portion of the platinum complex eventually remains bound to DNA, as after 24 h no significant fluorescence could be observed in the nucleus. Cisplatin-derived complexes with fluorescent tags afford a new insight into the cellular processing of these complexes and therefore may contribute to further unraveling of the mechanism of platinum antitumor complexes.

Antineoplastic Agents↗
PubMed2006

Structural insights into yeast septin organization from polarized fluorescence microscopy.

Septins are polymerizing GTPases that function in cortical organization and cell division. In Saccharomyces cerevisiae they localize at the isthmus between the mother and the daughter cells, where they undergo a transition from a non-dynamic hourglass-shaped assembly to two separate rings, at the onset of cytokinesis. Septins form filaments as pure protein and in vivo, but the filament organization within the hourglass and ring structures is controversial. Here, we use polarized fluorescence microscopy of orientationally constrained green fluorescent protein to determine septin filament organization and dynamics in living yeast. We found that the hourglass is made of filaments aligned along the yeast bud neck. During the transition from hourglass to rings the filaments rotate through 90 degrees in the membrane plane and become circumferential. These data resolve a long-standing controversy in the field and provide strong evidence that septins have a mechanical function in cell division.

Cell Cycle Proteins↗
PubMed1997

Detection of DNA breaks in apoptotic cells utilizing the DNA binding domain of poly(ADP-ribose) polymerase with fluorescence microscopy.

The DNA binding domain (DBD) of poly(ADP-ribose) polymerase (PARP) has proved to be a novel, highly sensitive probe for detecting DNA breaks in intact cells undergoing apoptosis. A recombinant peptide spanning the DNA binding domain of PARP was expressed, purified and used to detect DNA strand breaks in fixed cells. Fluorescence microscopy with this probe followed by detection with anti-PARP antisera initially revealed an increased binding following treatment of cells with DNA strand-breaking agents (such asN-methyl-N'-nitro-N-nitrosoguanidine) and, subsequently, using biotinylated PARP DBD, during the later stages of apoptosis in several cell systems, when internucleosomal strand breaks became evident. This procedure was found to be at least as sensitive and required fewer steps to detect DNA strand breaks than those utilizing Klenow incorporation of biotinylated nucleotides.

Apoptosis↗
PubMed2006

Dissecting elastic heterogeneity along DNA molecules coated partly with Rad51 using concurrent fluorescence microscopy and optical tweezers.

Nucleoprotein filament formation by recombinases is central to homologous recombination. To follow this process, we used fluorescent human Rad51 recombinase to visualize the interactions with double-stranded DNA (dsDNA). Fluorescence imaging revealed that Rad51 filament formation on dsDNA initiates from multiple nucleation points, resulting in Rad51-dsDNA nucleoprotein filaments interspersed with regions of bare DNA. The elastic properties of such heterogeneously coated DNA molecules were assessed by combining force-extension measurements using optical traps with fluorescence microscopy. This combination of single-molecule techniques allows discrimination of segments within an individual DNA molecule and determination of their elastic properties. The nonfluorescent zones of DNA-Rad51 constructs showed the well-known (over)stretching behavior of bare DNA. In contrast, the fluorescent, Rad51-coated zones did not overstretch and Rad51 remained stably bound in a structure that was approximately 50% longer than bare DNA. These results illustrate the power of adding sensitive fluorescence imaging to optical tweezers instrumentation.

DNA↗
PubMed1985

In vivo fluorescence microscopy of blood flow in mouse pancreatic islets: adrenergic effects in lean and obese-hyperglycemic mice.

The microcirculation in the islets of Langerhans was examined by fluorescence microscopy in living mice injected with fluorescent dextran. The islet capillary network was denser and more tortuously arranged in obese-hyperglycemic (ob/ob) mice than in lean controls. Injection of norepinephrine (0.5-4.0 micrograms/kg body wt) immediately led to a pronounced inhibition of islet blood flow in ob/ob mice. In experiments with lean mice less striking effects were seen. With as high a dose as 20 micrograms norepinephrine/kg body wt only a slight retardation and very brief stop of the flow occurred. The inhibition in ob/ob mice was blocked by phentolamine, indicating that the norepinephrine-induced inhibition was mediated by alpha-adrenoceptors. The alpha-2-adrenoceptor agonist, clonidine, had no effect on islet blood flow, suggesting that the effect of norepinephrine was due to alpha-1-adrenoceptor stimulation. It is concluded that in the living animal norepinephrine inhibits insulin secretion from the pancreas by a twofold mechanism involving inhibition of exocytosis (alpha-2-receptors on the beta-cells) as well as retardation of blood flow (alpha-1-receptors on blood vessels).

Animals↗
PubMed1990

Hydrolytic action of phospholipase A2 in monolayers in the phase transition region: direct observation of enzyme domain formation using fluorescence microscopy.

Phospholipase A2, a ubiquitous lipolytic enzyme highly active in the hydrolysis of organized phospholipid substrates, has been characterized optically in its action against a variety of phospholipid monolayers using fluorescence microscopy. By labeling the enzyme with a fluorescent marker and introducing it into the subphase of a Langmuir film balance, the hydrolysis of lipid monolayers in their liquid-solid phase transition region could be directly observed with the assistance of an epifluorescence microscope. Visual observation of hydrolysis of different phospholipid monolayers in the phase transition region in real-time could differentiate various mechanisms of hydrolytic action against lipid solid phase domains. DPPC solid phase domains were specifically targeted by phospholipase A2 and were observed to be hydrolyzed in a manner consistent with localized packing density differences. DPPE lipid domain hydrolysis showed no such preferential phospholipase A2 response but did demonstrate a preference for solid/lipid interfaces. DMPC solid lipid domains were also hydrolyzed to create large circular areas in the monolayer cleared of solid phase lipid domains. In all cases, after critical extents of monolayer hydrolysis in the phase transition region, highly stabile, organized domains of enzyme of regular sizes and morphologies were consistently seen to form in the monolayers. Enzyme domain formation was entirely dependent upon hydrolytic activity in the monolayer phase transition region and was not witnessed otherwise.

1,2-Dipalmitoylphosphatidylcholine↗