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PubMed2005

MR molecular imaging and fluorescence microscopy for identification of activated tumor endothelium using a bimodal lipidic nanoparticle.

In oncological research, there is a great need for imaging techniques that specifically identify angiogenic blood vessels in tumors on the basis of differences in the expression level of biomolecular markers. In the angiogenic cascade, different cell surface receptors, including the alphavbeta3-integrin, are strongly expressed on activated endothelial cells. In the present study, we aimed to image angiogenesis by detecting the expression of alphavbeta3 in tumor bearing mice with a combination of magnetic resonance imaging (MRI) and fluorescence microscopy. To that end, we prepared MR-detectable and fluorescent liposomes, which carry approximately 700 alphavbeta3-specific RGD peptides per liposome. RGD competition experiments and RAD-conjugated liposomes were used as controls for specificity. In vivo, both RAD liposomes and RGD liposomes gave rise to signal increase on T1-weighted MR images. It was established by the use of ex vivo fluorescence microscopy that RGD liposomes and RAD liposomes accumulated in the tumor by different mechanisms. RGD liposomes were specifically associated with activated tumor endothelium, while RAD liposomes were located in the extravascular compartment. This study demonstrates that MR molecular imaging of angiogenesis is feasible by using a targeted contrast agent specific for the alphavbeta3-integrin, and that the multimodality imaging approach gave insight into the exact mechanism of accumulation in the tumor.

Animals↗
PubMed1986

[Structural changes in the contractile proteins of muscle fiber studied by polarization ultraviolet fluorescence microscopy. IX. The effect of the pH and ionic strength of the solution on the conformational restructurings of F-actin induced by the binding of heavy meromyosin].

The dependence of F-actin conformational changes induced by the F-actin-HMM complex on pH and ionic strength was found by polarized ultraviolet fluorescence microscopy. It is discovered that pH affects sufficiently the cooperativity of F-actin structural changes, while the ionic strength affects their depth. The actomyosin complex was supposed to be at least in two structural states, differing in their orientation as well as in flexibility of F-actin monomers.

Actins↗
PubMed1989

Intravital fluorescence microscopy of endothelial cells on vascular grafts.

The ability to evaluate the extent of initial endothelial cell coverage on a vascular graft subsequent to an endothelial cell seeding technique would be desirable to substantiate the durability of the endothelial cell lining. To this end, we have evaluated the use of intravital fluorescence microscopy to assess human endothelial cell interaction with vascular grafts. Five fluorescent stains, mithramycin, Hoechst 33342, sulfofluorescein diacetate, Nile red, and rhodamine 123 were evaluated for their ability to fluorescently label human endothelial cells. The staining capability of each dye was also evaluated with respect to accuracy in determining seeded cell number and cell spreading. Of the stains evaluated, rhodamine 123 produced the most desirable characteristics. We observed excellent cell visualization after a 30-min incubation. Unlike the other four stains, rhodamine 123 exhibited a bright orange fluorescence emission at a 510-nm excitation wavelength while the underlying dacron or expanded polytetrafluoroethylene demonstrated minimal autofluorescence. Rhodamine 123 also exhibited no inhibitory effect on cell attachment to plastic or subsequent cell growth in culture. Intravital fluorescence microscopy could be easily utilized in the operating room to visualize part or all of an endothelial cell-seeded graft prior to implantation and would permit a quantitative as well as qualitative evaluation of the seeding process. Since intravital fluorescence imaging does not require tissue fixation, the same surface as that evaluated for seeding efficiency can be directly implanted.

Blood Vessels↗
PubMed1996

Measurements by fluorescence microscopy of the time-dependent distribution of meso-tetra-hydroxyphenylchlorin in healthy tissues and chemically induced "early" squamous cell carcinoma of the Syrian hamster cheek pouch.

The influence of the time interval between dye administration and detection by fluorescence microscopy was assessed in "early" squamous cell carcinomas of the cheek pouch mucosa and different healthy tissues of the Syrian hamster. Following intracardiac injection of 0.15 mg (kg bodyweight)-1 of meso-tetra-hydroxyphenylchlorin (m-THPC), groups of three animals were sacrificed at different time intervals up to 30 days. A group of three non-injected animals was used to detect the endogene fluorescence of the corresponding normal tissues for autofluorescence subtraction. The following excised organs (oesophagus, trachea, liver, spleen, kidney, skin, striated muscle, healthy and tumoral cheek pouch mucosae) were fast frozen in liquid nitrogen and stored at -70 degrees C for fluorescence microscopy. The results show significant differences in the detectable m-THPC levels in different tissue layers (for instance, the epithelia and muscle of the oesophagus, trachea and cheek pouch) at different time intervals. These data indicate that pharmacokinetic studies may be useful for selecting the optimal time for the photodetection and phototherapy of cancer.

Animals↗
PubMed1966

Modifications in the fluorescence microscopy technique as applied to identification of acid-fast bacilli in tissue and bacteriological material.

The principles and techniques involved in fluorescence microscopy as related to the demonstration of acid-fast bacilli are discussed. The auramine-rhodamine staining method, for both tissue and bacteriological preparations, is described as well as the necessary equipment for both microscopy and photomicroscopy. Fluorescence staining is no more difficult nor time consuming than a Ziehl-Neelsen stain yet it is more effective in demonstrating acid-fast organisms.

Bacillus↗
PubMed2000

Fluorescence Microscopy Study of Heterogeneity in Polymer-supported Luminescence-based Oxygen Sensors.

Despite the great potential of fluorescence microscopy, its application to date has largely been in the study of biological specimens. It will be shown that conventional fluorescence microscopy provides an invaluable tool with which to study the photophysics of polymer-supported luminescence-based oxygen sensors. The design of the imaging system, the measurement methods, and the data analysis used in the investigation of sensor systems are described. Fluorescence microscopic images of sensor films in which microheterogeneous regions exhibiting enhanced luminescence intensity and poorer oxygen quenching relative to the bulk response are shown. This is the first direct evidence that sensor molecules in various domains of the polymer support can exhibit different oxygen quenching properties. It will be shown that µ- and nano-crystallization of the sensor molecule are the probable source of both the observed heterogeneous microscopic responses and the microscopic and macroscopic nonlinear Stern-Volmer plots. The implications of these results in the rational design of luminescence-based oxygen sensors are discussed.

Journal Article↗
PubMed2005

Development of a coculture system and use of confocal laser fluorescent microscopy to study human microvascular endothelial cell and mural cell interaction.

In the present study, human myometrial microvascular endothelial cells (HMMEC) were cocultured with human vascular smooth muscle cells (VSMC) labeled with fluorescent dyes to examine their morphological interaction using confocal laser fluorescent microscopy. HMMEC and VSMC labeled with fluorescent green and red dyes, respectively, attached to opposite sides of polyethyleneterephthalate membranes and remained viable for up to 96 h. In defined medium, 5%+/-3% of the VSMC cytoplasmic processes and 71%+/-17% of the HMMEC processes extended completely across the 13 microm thickness of the transmembrane. However, 41%+/-21% of the VSMC projections and 10%+/-3% of the HMMEC processes that traversed the membrane made contact with the opposing cell type. In cocultures incubated with angiopoietin-1 (Ang-1), although the number of VSMC or HMMEC projections was not significantly increased, the number of VSMC extending across the membrane and making contact with HMMEC was increased (P<0.05) to 88%+/-2%. The results of the current study demonstrate that coculture of fluorescent-labeled HMMEC and VSMC on a semipermeable transmembrane coupled with confocal laser fluorescent microscopy provides an in vitro experimental model to study morphological association of microvascular endothelial cells with mural cells. We propose that this system will greatly facilitate study of remodeling of the microvasculature in various organ systems.

Angiopoietin-1↗
PubMed1993

Use of the UV ParaLens adapter as an alternative to conventional fluorescence microscopy for detection of Pneumocystis carinii in direct immunofluorescent monoclonal antibody-stained pulmonary specimens.

The UV ParaLens light microscope adapter offers a useful and cost-effective alternative to conventional fluorescence microscopy for Pneumocystis carinii identification, particularly in AIDS patients. In a blinded study, in which 153 pulmonary specimens were examined for P. carinii by direct immunofluorescence, 40 of 42 specimens positive by fluorescence microscopy were also positive by ParaLens. No false positives were observed.

Antibodies, Fungal↗
PubMed2005

Observation of the main phase transition of dinervonoylphosphocholine giant liposomes by fluorescence microscopy.

The phase heterogeneity of giant unilamellar dinervonoylphosphocholine (DNPC) vesicles in the course of the main phase transition was investigated by confocal fluorescence microscopy observing the fluorescence from the membrane incorporated lipid analog, 1-palmitoyl-2-(N-4-nitrobenz-2-oxa-1,3-diazol)aminocaproyl-sn-glycero-3-phosphocholine (NBDPC). These data were supplemented by differential scanning calorimetry (DSC) of DNPC large unilamellar vesicles (LUV, diameter approximately 0.1 and 0.2 microm) and multilamellar vesicles (MLV). The present data collected upon cooling reveal a lack of micron-scale gel and fluid phase coexistence in DNPC GUVs above the temperature of 20.5 degrees C, this temperature corresponding closely to the heat capacity maxima (T(em)) of DNPC MLVs and LUVs (T(em) approximately 21 degrees C), measured upon DSC cooling scans. This is in keeping with the model for phospholipid main transition inferred from our previous fluorescence spectroscopy data for DMPC, DPPC, and DNPC LUVs. More specifically, the current experiments provide further support for the phospholipid main transition involving a first-order process, with the characteristic two-phase coexistence converting into an intermediate phase in the proximity of T(em). This at least macroscopically homogenous intermediate phase would then transform into the liquid crystalline state by a second-order process, with further increase in acyl chain trans-->gauche isomerization.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1977

Smear results in the diagnosis of mycobacterioses using blue light fluorescence microscopy.

Examination of 6,880 sputum specimens from untreated patients disclosed that 3.1% were positive for mycobacteria by fluorescence microscopy, and 92.5% of these has positive cultures. There was a positive correlation between the number of organisms seen on smear and growth on culture. All specimens with positive smears and negative cultures contained rare or few acid-fast bacilli on the smear. Eighty-two percent of the specimens with positive cultures and negative smears yielded less than 25 colonies, whereas there were greater than 25 colonies from 93% of the specimens with positive smears and cultures. In a low prevalence, general hospital population, the primary acid-fast smear continued to be a reliable diagnostic tool.

Bacteriological Techniques↗
PubMed2004

Imaging the activity and localization of single voltage-gated Ca(2+) channels by total internal reflection fluorescence microscopy.

The patch-clamp technique has enabled functional studies of single ion channels, but suffers limitations including lack of spatial information and inability to independently monitor currents from more than one channel. Here, we describe the use of total internal reflection fluorescence microscopy as an alternative, noninvasive approach to optically monitor the activity and localization of multiple Ca(2+)-permeable channels in the plasma membrane. Images of near-membrane Ca(2+) signals were obtained from >100 N-type channels expressed within restricted areas (80 x 80 micro m) of Xenopus oocytes, thereby permitting simultaneous resolution of their gating kinetics, voltage dependence, and localization. Moreover, this technique provided information inaccessible by electrophysiological means, demonstrating that N-type channels are immobile in the membrane, show a patchy distribution, and display diverse gating kinetics even among closely adjacent channels. Total internal reflection fluorescence microscopy holds great promise for single-channel recording of diverse voltage- and ligand-gated Ca(2+)-permeable channels in the membrane of neurons and other isolated or cultured cells, and has potential for high-throughput functional analysis of single channels.

Animals↗
PubMed1979

Rapid detection of malaria and other bloodstream parasites by fluorescence microscopy with 4'6 diamidino-2-phenylindole (DAPI).

DAPI is a fluorescent dye which appears to complex specifically with DNA. We have used this probe to detect and identify malarial infections by fluorescence microscopy. Experiments were conducted using Plasmodium berghei yoeli--infected mouse blood, P. lophurae--infected duck blood, and P. vivax--infected human blood. Infected avian blood was used to detect parasites within nucleated erythrocytes. Control blood smears from uninfected hosts revealed fluorescence only in the leukocytes of mammalian blood or in nuclei of leukocytes and erythrocytes of avian blood. Cytoplasmic staining of red blood cells was absent in all controls. In contrast, the cytoplasm of infected red blood cells was stippled with fluorescence centers. Ring forms, trophozoites, segmenters, and merozoites frequently were observed. This simple procedure can be applied directly to routine clinical analysis, as well as experimental procedures, DAPI can also be used to stain other parasites, including nuclei in microfilariae.

Amidines↗
PubMed1989

Fluorescence microscopy of DES-induced morphologic transformation in unfixed, cultured cells.

Cultured baby Syrian hamster kidney cells (BHK 21), synchronized with hydroxyurea (HU), were treated with varying amounts of the carcinogen diethylstilbestrol (DES) during the first mitosis. DES-induced morphologic transformation was assessed by fluorescence microscopy of acridine orange, supravitally stained cells. Traditional karyotyping techniques were used to monitor DES-induced aneuploidy in parallel cell cultures. A total of 618 photomicrographs were obtained by systematic manual scanning of three replicate experiments each using three different DES treatment levels and a no DES control. These photomicrographs were randomized and graded blind by three independent observers. Each photomicrograph was graded for cellular morphologic transformation and a judgement as to whether DES treatment had been used. A definite dose-response relationship for both morphologic transformation, as well as for aneuploidy was observed. These findings portend a significance for use of fluorescence microscopy of morphologic transformation of unfixed supravitally stained mammalian cells, for the rapid assessment (24 h or less) of compounds that promote aneuploidization and carcinogenesis.

Acridine Orange↗
PubMed1978

[Study of structural changes in muscle fiber contractile proteins using polarization ultraviolet fluorescent microscopy. IV. Several features of the conformational changes in F-actin during muscle fiber relaxation].

Using the polarized ultraviolet fluorescent microscopy in the presence of a fluorescence quencher (acrylamide, NO3-) it has been shown that actin tryptophanyls are oriented with their short axes perpendicular to the long axis of the thin filament. Fluorophores in hardly accessible sites of the macromolecule have a more pronounced anisotrophy of orientation in comparison with those in the protein sites easily available by a fluorescence quencher. During the muscle fiber relaxation, conformational changes of F-actin take place which embrace seemingly the sites of subunits, close to the surface of macromolecule, and make difficult the penetration of univalent ions and neutral molecules deep into the protein macromolecule. Some connection between conformational changes of the surface areas of F-actin subunits and the actin incapability of combining with HMM in the presence of ATP is assumed.

Acrylamides↗
PubMed1988

Distribution of microtubules and other cytoskeletal filaments during myotube elongation as revealed by fluorescence microscopy.

Distribution of microtubules and other cytoskeletal filaments in growing skeletal muscle cells (myotubes) was studied in vitro by fluorescence microscopy using fluorescin-labeled antibodies and phalloidin, a specific antiactin drug. In the distal elongating tips of myotubes, microtubules were the major cytoskeletal elements; actin and intermediate filaments were much less abundant. On the other hand, colcemid- and nocodozole-treatments caused disruption of microtubles and also prompt retraction of growth tips to form myosacs, a type of deformed myotube. Actin filaments remained unaffected during the retraction. The difference in the distribution of the 3 cytoskeletal filaments in the region of growth tips was most remarkable in the case of those myotubes in the process of recovery from myosacs. In an early phase of recovery, the cellular processes extending from myosacs were enriched with both microtubules and intermediate filaments, but not with actin filaments. Later, when the processes became further developed, intermediate filaments were scarce at the extreme ends. Fluorescein-labeled actin introduced by a micro-injection method was minimally incorporated into filaments in the cellular processes. We conclude that microtubules make up the cytoskeletal element which is most responsible for elongation or spreading of growth tips of myotubes in vitro.

Actins↗
PubMed2005

Waveguide excitation fluorescence microscopy: a new tool for sensing and imaging the biointerface.

A novel biosensing and imaging technique, the waveguide excitation fluorescence microscope, has been developed for the dynamic and quantitative investigation of bio-interfacial events in situ, ranging from ligand-receptor binding to focal adhesion formation in cell-surface interactions. The technique makes use of the evanescent field created when light travels in a mono-mode, planar optical waveguide to excite fluorescence in the near interface region. Advantages of the technique include high target sensitivity for fluorescence detection (femtomolar range), high surface specificity (ca. 100 nm perpendicular to the waveguide), large area analysis with submicron resolution, 'built-in' calibration of fluorescent light gain, and the capability to perform multi-colour imaging in situ and in real time. In this work, the sensitivity of the system has already been demonstrated through dynamic measurements of the streptavidin-biotin binding event to below 20 pM concentrations, signal to noise comparisons with conventional fluorescence microscopy have shown more than a 10-fold improvement, and surface specificity of the technique has also been illustrated in a comparison of fibroblast focal adhesion images. Thus, this new tool can be used to illuminate processes occurring at the interface between biology and synthetic surfaces in a unique manner.

Biosensing Techniques↗
PubMed1986

Testicular microcirculation in the rat studied by videophotometric capillaroscopy, fluorescence microscopy and laser Doppler flowmetry.

Testicular capillary blood flow was studied in rats using laser Doppler flowmetry, in vivo fluorescence microscopy and videophotometric capillaroscopy. All the methods revealed rhythmical oscillations in testicular microcirculation with a periodicity of 4-10 c.p.m. In arterioles, capillaries and small post-capillary vessels, periods of continuous blood flow alternated with periods of no or very low flow. No visible leakage of dextran-150 was observed from the testicular blood vessels. Four, 8 and 16 h after an s.c. injection of 200 IU hCG the blood flow was continuous and there was leakage of dextran-150 from the microvessels to the interstitial tissue. Twenty-four and 32 h after hCG the blood flow pattern was again rhythmical, and at 32 h there was no leakage of dextran-150. This suggests that hCG induces changes in blood flow and transvascular fluid exchange in the testis, perhaps by altering smooth muscle activity at the arteriolar-level.

Animals↗