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Results for “Confocal microscopy”

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PubMed2007

Atomic force and confocal microscopy for the study of cortical cells cultured on silicon wafers.

The primary cortical cells were selected as a model to study the adherence and neural network development on chemically roughened silicon substrates without any coatings using confocal laser scanning microscopy (CLSM) and atomic force microscopy (AFM). The silicon substrates have a nano-range roughness (RMS) achieved by chemical etching using hydrofluoric (HF) acid. After 7 days of culturing, the neurons were observed to connect together and form dense neural networks. Furthermore, AFM results revealed that some porous structures at a few micrometer range existed between the neuron cells and the silicon substrates. It is suggested that the porous structures are made of extracellular matrix (ECM) components and play an important role in the neuronal adhesion and neurite outgrowth on the inert silicon wafers.

Animals↗
PubMed2005

Microstructure of acid-induced caseinate gels containing sucrose: quantification from confocal microscopy and image analysis.

We investigate the effect of sucrose on the microstructure of sodium caseinate gels induced by acidification. The average pore size and the fractal properties of two-dimensional slices of the gels are studied using confocal scanning laser microscopy and image analysis. The addition of sucrose promotes stronger and more fine-stranded gels while re-arrangements of the network tend to be prevented. Whereas the fractal dimension itself is not sensitive to changes in the gel microstructure upon addition of sucrose, the maximum cut-off distance, up to which fractal scaling behaviour applies, is substantially reduced, as is the average pore size. The overall microstructural changes seem to be consistent with previous rheological and light scattering studies of the same system.

Acids↗
PubMed1990

Subcellular calcium transients visualized by confocal microscopy in a voltage-clamped vertebrate neuron.

Confocal laser-scanned microscopy and long-wavelength calcium (Ca2+) indicators were combined to monitor both sustained and rapidly dissipating Ca2+ gradients in voltage-clamped sympathetic neurons isolated from the bullfrog. After a brief activation of voltage-dependent Ca2+ channels, Ca2+ spreads inwardly, and reaches the center of these spherical cells in about 300 milliseconds. Although the Ca2+ redistribution in the bulk of the cytosol could be accounted for with a radial diffusion model, local nonlinearities, suggesting either nonuniform Ca2+ entry or spatial buffering, could be seen. After electrical stimulation, Ca2+ signals in the nucleus were consistently larger and decayed more slowly than those in the cytosol. A similar behavior was observed when release of intracellular Ca2+ was induced by caffeine, suggesting that in both cases large responses originate from Ca2+ release sites near or within the nucleus. These results are consistent with an amplification mechanism involving Ca2(+)-induced Ca2+ release, which could be relevant to activity-dependent, Ca2(+)-regulated nuclear events.

Animals↗
PubMed2000

Methods for imaging the structure and function of living tissues and cells: 3. Confocal microscopy and micro-radiology.

This review article looks at the development of confocal imaging technology, with emphasis on its abilities to overcome some of the problems of imaging life processes, particularly in the intact organ or animal. A brief summary of three promising micro-imaging modalities is provided (which are microscopical analogues of conventional radiological techniques) with a bibliography for the interested reader to pursue.

Animals↗
PubMed2000

Use of confocal microscopy to detect Salmonella typhimurium within host cells associated with Spv-mediated intracellular proliferation.

The major limitation in histological examination of orally inoculated mice of Salmonella typhimurium has been the difficulty of attaining high enough levels for immunochemical detection. This problem has been solved by the use of confocal laser scanning microscopy (CLSM) analysis, which allows detection of bacteria in the immunostained sections of mouse spleens at a minimum rate of approximately 1000 colony-forming units (cfu)/spleen. Here, we demonstrate that over 80% of salmonellae of the wild type of S. typhimurium were detected intracellularly within Mac-1 positive cells by the CLSM analysis of immunostained sections from spleens in orally or subcutaneously inoculated mice. Only 40% of salmonellae of the spv -deleted strain were detected inside Mac-1 positive cells. These data suggest that the spv genes play a key role in intracellular proliferation within phagocytes in the mouse spleen.

Animals↗
PubMed2005

A procedure to determine the correct thickness of an object with confocal microscopy in case of refractive index mismatch.

A difference in refractive index (n) between immersion medium and specimen results in increasing loss of intensity and resolution with increasing focal depth and in an incorrect axial scaling in images of a confocal microscope. Axial thickness measurements of an object on such images are therefore not exact. The present paper describes a simple procedure to determine the correct axial thickness of an object with confocal fluorescence microscopy. We study this procedure for a specimen that has a higher refractive index than the immersion medium and with a thickness up to 100 microm. The measuring method was experimentally tested by comparing the thickness of polymer layers measured on axial images of a confocal microscope in case of a water-polymer mismatch to reference values obtained from an independent technique, i.e. scanning electron microscopy. The case when the specimen has a lower refractive index than the immersion medium is also shown by way of illustration. Measured thickness data of a water layer and an oil layer with the same actual thickness were obtained using an oil-immersion objective lens with confocal microscopy. Good agreement between theory and experiment was found in both cases, consolidating our method.

Journal Article↗
PubMed1997

Effect of low concentration of epinephrine on human platelet aggregation analyzed by particle counting method and confocal microscopy.

The effect of a physiologic concentration of epinephrine (Ep) on platelet activation was studied by using a novel method that simultaneously measures platelet aggregation by changes in light transmission and counts particles of various sizes by using light scattering. Detailed morphologic changes associated with activation process were studied by using confocal laser scanning microscopy (CLSM). A low concentration of Ep (20 nmol/L) corresponding to a high physiologic concentration triggered the formation of small platelet aggregates (diameter 7 to 30 microm) without any change in light transmission. The redistribution of filamentous actin (F-actin) and the expression of activated glycoprotein IIb-IIIa complex (GPIIb-IIIa), detected by PAC-1 binding, were also observed in the platelets comprising the small aggregates. Attempts were then made to detect changes in cytoplasmic ionized Ca2+ ((Ca2+)i) in individual platelets involved in the aggregate formation by CLSM with fluo-3, a Ca2+-indicating dye. Ep caused a weak (Ca2+)i increase in some individual platelets involved in the formation of small aggregates. This (Ca2+)i increase was associated with platelet aggregation, because no (Ca2+)i rise was detected in single platelets. Furthermore, platelets stimulated by Ep in the presence of RGDS or Ro 44-9883, a GPIIb-IIIa antagonist, did not form small aggregates or trigger a (Ca2+)i rise. Prior incubation with low concentrations of Ep (20, 100 nmol/L) enhanced the initial formation of small (diameter 7 to 30 microm), medium (diameter 30 to 50 microm), or large (diameter 50 to 70 microm) aggregates induced by a subthreshold concentration of adenosine diphosphate (0.25 micromol/L) as determined by the particle counting method. However, no apparent synergistic (Ca2+)i increase was observed in platelets involved in aggregate formation. From these observations the following conclusions have been reached. (1) A high physiologic concentration of Ep (20 nmol/L) is capable of triggering the formation of small aggregates, resulting in the redistribution of F-actin and the expression of activated GPIIb-IIIa complex. (2) An increase in (Ca2+)i is observed in platelets comprising the small aggregates. This increase is not related to the binding of Ep to its receptor but most likely is triggered by platelet-platelet association. (3) The characteristic potentiating effect of Ep is not due to the synergistic increase in (Ca2+)i.

Actins↗
PubMed1995

Basic science and applications of in vivo microscopy.

Confocal microscopy creates a scanned image from a point light source and point detection or a scanning slit to remove scattered light and improve optical resolution. This also results in optical sectioning of tissues. These capabilities can be employed to image structures in the human cornea, in vivo, both for research and for the diagnosis and treatment of human disease. Optical sections, when recombined, can lead to three-dimensional reconstructions from which very useful information is obtained. Investigators have found keratocyte density decreases from anterior to posterior in the stroma of the rabbit cornea. Surface epithelial desquamation can also be studied and the effects of contact lens use can be demonstrated. The instrument is also useful for diagnosing and guiding therapy for some human diseases such as Acanthamoeba keratitis. Colonies of bacteria may also be observed and treatment evaluated in patients with infectious crystalline keratopathy. Confocal microscopy can also image the retina.

Contact Lenses↗
PubMed1995

Confocal microscopy analysis of NPY and TH immunoreactivities in the hypothalamo-hypophysial system of the frog.

Co-expression of tyrosine hydroxylase (TH) and neuropeptide Y (NPY) in local circuits innervating the hypothalamo-pituitary complex of the green frog, Rana ridibunda, was investigated using simultaneous double immunohistochemical technique, aided by dual-channel confocal laser scanning microscopy. NPY and TH immunoreactivities were observed co-occurring within a discrete neuronal population located in the suprachiasmatic region. In other hypothalamic areas, NPY-immunoreactive (IR) perikarya were generally codistributed, but distinct from TH-IR cells. In the adenohypophysial pars intermedia, the overlap between the two markers was partial, demonstrating the existence of multiple neuronal sources for the inputs to the gland.

Animals↗
PubMed2002

Employment of confocal microscopy for the dynamic visualization of domes in intact epithelial cell cultures.

Many epithelial cells cultured on plastic ware form domes, fluid-filled localized raisings of the cell monolayer. Domes are due to active vectorial ion transport and their presence demonstrates the maintenance of a differentiated polarized phenotype and of tight junctional complexes. Through a confocal laser microscope equipped with a special flow chamber, intact domes were evaluated in real time for prolonged experimental periods. Both in CAPAN-1 pancreatic duct adenocarcinoma cells and in renal tubular LLC-PK1 cells, vertical sections of calcein-loaded cultures provided a clear visualization of dome outlines during the slow deflation induced by specific agonists (respectively, 1 microM secretin or 10 microM vasopressin). Section series of calcein-loaded domes were used for three-dimensional reconstructions. In CAPAN-1 cultures, cell depolarization induced by secretin was detected with the potentiometric dye bis-oxonol. In the same cells pyranine, a fluid phase marker that is cell impermeant, visualized dome compartment and paracellular pathways, also providing an evaluation of dome fluid pH. Confocal laser scanning microscopy of domes represents a convenient device for the functional assessment of living epithelial cells.

Arylsulfonates↗
PubMedDate not supplied

Two- and three-dimensional distributions of opioid receptors on NG108-15 cells visualized with the aid of fluorescence confocal microscopy and anti-idiotypic antibodies.

Polyclonal anti-idiotypic antibodies previously shown to be specific for mu- and delta-opioid receptor subclasses on rat brain membrane preparations using radioreceptor assays have been labeled with fluorescent conjugates of avidin. The resulting complexes have been used to study the distribution and properties of opioid receptors on living and fixed NG108-15 cells using normal fluorescence, and confocal fluorescence, microscopy. From the confocal data three-dimensional views of immunofluorescent distributions have been obtained using a computer transformation of the data. From experiments involving co-incubation with media containing naloxone, selective opioid peptides and stereoisomeric synthetic opioid analogs as blocking agents, the specificity and stereospecificity of immunofluorescent binding is reported. The effects on immunofluorescent binding by co-incubation with media containing a sulfhydryl reagent, 100 mM-NaCl and sodium azide are also reported. The results have implications concerning the use of immunoprecipitation in the biochemical isolation of the receptors. Finally, the confocal two-dimensional sections and two-dimensional reconstructions obtained from fixed cells show internal immunofluorescence in the form of cytoplasmic clusters. The reagents and methods should be applicable to visualizing native opioid receptor distributions on neurons.

Animals↗
PubMed2001

Differences in the intracellular distribution of acid-sensitive doxorubicin-protein conjugates in comparison to free and liposomal formulated doxorubicin as shown by confocal microscopy.

PURPOSE: To investigate differences in the cellular uptake and intracellular distribution of protein-bound doxorubicin in comparison to free doxorubicin and a liposomal formulation (CAELYX) METHODS: LXFL 529 lung carcinoma cells were incubated with an acid-sensitive transferrin and albumin conjugate of doxorubicin, a stable albumin doxorubicin conjugate, and free and liposomal doxorubicin for up to 24 h. The uptake of doxorubicin was detected with confocal laser scanning microscopy (CLSM). To investigate the intracellular localization of the anticancer drug, lysosomes, Golgi apparatus, and mitochondria were also stained by various organelle-specific fluorescent markers. In vitro efficacy of the doxorubicin derivatives was examined with the BrdU incorporation assay. RESULTS: The acid-sensitive albumin and transferrin doxorubicin conjugates showed enhanced cytotoxicity in comparison to liposomal doxorubicin, whereas the stable albumin-doxorubicin conjugate showed only marginal activity. Of all compounds tested, doxorubicin showed the highest cytotoxicity. CLSM studies with specific markers for lysosomes, mitochondria, and the Golgi apparatus demonstrated that protein-bound doxorubicin or liberated doxorubicin was accumulated in the mitochondria and Golgi compartments, but not in the lysosomes after 24 h. Free doxorubicin showed a time-dependent intracellular shift from the nucleus to the mitochondria and Golgi apparatus. Fluorescence resulting from incubation with CAELYX was primarily detected in the nucleus. CONCLUSIONS: Our results indicate that other organelles in addition to the cell nucleus are important sites of accumulation and interaction for protein-bound doxorubicin or intracellularly released doxorubicin as well as for free doxorubicin.

Antineoplastic Agents↗
PubMed2006

Hermaphrodites and supernumerary testicular lobes in Schistosoma mansoni (Trematoda: Schistosomatidae) analyzed by brightfield and confocal microscopy.

Schistosoma mansoni belongs to the dioecious Schistosomatidae. The occasional observation of males with rudimentary female characteristics is thought to attest the hermaphroditic roots of this parasite. Supernumerary testicular lobes also are recurrently seen in this helminth, but their morphology and origin are elusive. Here, we investigated the morphology of the supernumerary lobes from 15 S. mansoni males and similar structures of 2 females in whole mounts by brightfield and confocal laser scanning microscopy. The follicles in the females were not regarded as testicular lobes, but 1 male had a rudimentary ovary with a residual oviduct posterior to the normal set of testicular lobes and was considered hermaphroditic. In addition, 6 other males contained supernumerary lobes that enclosed fully matured oocytes in variable amounts. In the remaining 8 males, no female characteristics could be seen, and the lobes showed morphologically a more testicular aspect, although this morphology was only confirmed in 1 male where spermatozoids were detected. The process underlying the expression of supernumerary testes or lobes with oocytes is similar and is, at least in part, induced by the host.

Animals↗
PubMed1999

Intravital laser confocal microscopy of pulmonary edema resulting from intestinal ischemia-reperfusion injury in the rat.

OBJECTIVE: To observe pulmonary edema resulting from intestinal ischemia-reperfusion injury. We used a newly developed laser confocal microscope to observe the subpleural capillary network and the superficial alveoli under intravital conditions, and created three-dimensional images of the pulmonary microcirculation to analyze the time course and spatial pattern of pulmonary exudative changes during intestinal ischemia-reperfusion injury in vivo. DESIGN: Prospective, randomized, unblinded study. SETTING: Laboratory of a university hospital. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: The rats were injected intravenously with bovine serum albumin labeled with fluorescein isothiocyanate and subjected to 60 mins of intestinal ischemia, followed by 180 mins of reperfusion. During mechanical ventilation, the upper lobe of the right lung was examined in the intravital state using a high-speed confocal fluorescence microscope. MEASUREMENTS AND MAIN RESULTS: Interstitial edema and alveolar leakage were recognized as changes of interstitial fluorescence in the subpleural capillary network and as changes of alveolar fluorescence in the alveolar cross-sectional view. Although exudative changes in the interstitium and alveoli were observed during intestinal ischemia, there was a marked increase in both interstitial edema and alveolar leakage after intestinal reperfusion. CONCLUSION: We observed pulmonary edema under intravital conditions and demonstrated the utility of a newly developed laser confocal microscope. This system not only enabled us to analyze the development of pulmonary edema three-dimensionally, but also allowed us to evaluate the pulmonary microcirculation.

Analysis of Variance↗
PubMed2004

Measuring membrane potential and electric field of brainstem neurons in vitro by confocal microscopy.

A method of measuring transmembrane potential and electric field of neural cells cultured in vitro is described in this paper. The high resolution and scanning speed of the method make it considerable to be used to observe the viability of the neurons cultured on opaque substrate. Rhodamine 123 was used to stain the cells in order to display different intensity corresponding to transmembrane potential. The fluorescence data were collected by confocal laser scanning microscopy (CLSM). Then the data were processed to create the graphs of transmembrane potential and electric field. This is the first paper describes a reliable method for three-dimensional visualization of potential voltage of neurons at the best of our knowledge.

Animals↗
PubMed1998

MDCK cell cultures as an epithelial in vitro model: cytoskeleton and tight junctions as indicators for the definition of age-related stages by confocal microscopy.

PURPOSE: Madin Darby Canine Kidney (MDCK) cells were grown in culture, and age-related morphological changes in the cytoskeleton and tight junction (TJ) network were used to define stages in view of establishing an optimal in vitro model for the epithelial barrier. METHODS: Growth curves and transepithelial electrical resistance (TEER) were determined, and the cytoskeleton (actin, alpha-tubulin, vimentin) and TJ (Zonula occludens proteins ZO1, ZO2) were investigated with immunofluorescent methods by confocal laser scanning microscopy (CLSM) and digital image restoration. RESULTS: TEER measurements indicated that TJ were functional after one day. Values then remained constant. Four morphological stages could be distinguished. Stage I (0-1 day): Sub confluent cultures with flat cells; TJ established after cell-to-cell contacts are made. Stage II (2-6 days): Confluent monolayers with a complete TJ network, which remains intact throughout the later stages. Stage III (7-14 days): Rearrangement in the cytoskeleton; constant cell number; volume and surface area of cells reduced (cobble-stone appearance). Stage IV (> or = 15 days): Dome formation, i.e. thickening and spontaneous uplifting of the cell monolayer. CONCLUSIONS: Based on the structural characteristics of stage III cell cultures, which are closest to the in vivo situation, we expect them to represent an optimal in vitro model to study drug transport and/or interactions with drugs and excipients.

Animals↗
PubMed2006

Stored human septal chondrocyte viability analyzed by confocal microscopy.

OBJECTIVES: To analyze the effects of prolonged storage time, at warm and cold temperatures, on the viability of human nasal septal chondrocytes and to understand the implications for tissue engineering of septal cartilage. DESIGN: Basic science. SUBJECTS: Septal cartilage was obtained from 10 patients and placed in bacteriostatic isotonic sodium chloride solution. Four specimens were kept at 23 degrees C, and 4 were kept at 4 degrees C. The viability of the chondrocytes within the cartilage was assessed using confocal laser scanning microscopy every 5 days. The 2 other specimens were assessed for viability on the day of harvest. RESULTS: Viability on the day of harvest was 96%, implying minimal cell death from surgical trauma. After 1 week, cell survival in all specimens was essentially unchanged from the day of harvest. At 23 degrees C, the majority (54%) of cells were alive after 20 days. At 4 degrees C, 70% of cells survived 1 month and 38% were alive at 2 months. Qualitatively, chondrocytes died in a topographically uniform distribution in warm specimens, whereas cold specimens displayed a more irregular pattern of cell death. CONCLUSION: Septal chondrocytes remain viable for prolonged periods when stored in simple bacteriostatic isotonic sodium chloride solution, and such survival is enhanced by cold storage.

Adult↗
PubMed2002

Influence of refractive-index mismatch in high-resolution three-dimensional confocal microscopy.

The effects of the refractive-index mismatch in confocal laser scanning microscopy were extensively studied. The axial aberration induced in the case of fluorescent microspheres was measured. The data were used to take into account the mismatch-induced aberrations and to consider object-size influence. Then we focused on the effect of refractive-index mismatch on the effective system's point-spread function under different mismatch conditions and on depth of focusing. We experimentally verified that the peak of the point-spread function intensity profile decreases and the point-spread function itself progressively broadens as a function of the combined effect of the refractive-index mismatch and of the penetration depth, leading to a worsening of the system's overall performances. We also performed these same measurements by embedding subresolution beads in an oocyte's cytoplasm, which can be considered a turbid medium. We found evidence consistent with the previously developed theoretical model; in particular we found a strong dependence of the intensity peak on the focusing depth.

Journal Article↗