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PubMedDate not supplied

In vitro histoculture of human tumors with fluorescent dye end-points measured by confocal microscopy: high correlation of in vitro and in vivo chemosensitivity.

Tumors from 40 patients and 7 established human xenograft tumor lines were grown in three-dimensional histoculture. A Viable-Cell-Index (VCI) based on fluorescent dyes and Growth Fraction Index (GFI) based on [3H]thymidine incorporation were measured by confocal microscopy and histological autoradiography, respectively, after treatment with cytotoxic agents. Chemosensitivity in vitro with the two methods was correlated with chemosensitivity of the same set of human xenografted tumor lines grown in nude mice. The percent accuracy of in vitro to in vivo correlation with VCI (73%) was higher than GFI (63%). The number of false positives with VCI was 12.1% (4/33), and with GFI was 31.3% (10/32). The results thus indicated that in vitro histoculture with fluorescent vital-dye end-points to measure cell viability is of potential use to determine tumor chemosensitivity.

Animals↗
PubMed2005

Musculoskeletal responses of 2-year-old Thoroughbred horses to early training. 8. Quantitative back-scattered electron scanning electron microscopy and confocal fluorescence microscopy of the epiphysis of the third metacarpal bone.

AIM: To characterise and explain the increase in density evident by computerised tomography (CT) and radiography in companion studies as a response to training, in bone in the palmar and dorsal regions of the condyles of the third metacarpal bone (Mc3) of 2-year-old Thoroughbred horses. METHODS: Compositional back-scattered electron (BSE) imaging in scanning electron microscopy (SEM) and confocal scanning laser microscopy (CSLM) were conducted on polymethyl methacrylate (PMMA)-embedded mediolateral slices of the right distal Mc3 from seven 2-year-old Thoroughbred horses trained on a racetrack and seven untrained horses kept at pasture. One left Mc3 from each group was studied in transverse section planes. This study focussed on regions of Mc3 found to differ in density between the trained and untrained horses in companion studies using CT and radiography. RESULTS: The increase of bone density in the condyles of Mc3 in trained horses compared with untrained horses occurred, without prior osteoclastic resorption, via the deposition of new bone on pre-existing internal surfaces. Within prior marrow spaces of cancellous bone, there was also rapid formation of immature strands and fronds of bone which were more cellular and mineralised, and more lamellar bone tissue was deposited on these new scaffolding elements in the trained horses. Both resulted in increased bone volume fraction (BVF). The microscopic mineralisation density of the bulk of the new tissue was lower than in pre-existing bone, and CT and radiography underestimated the increase in BVF. The new tissue was thus probably less stiff at the microscopic scale than pre-existing bone, though its addition would stiffen the global structure. CONCLUSIONS: In Mc3 of all the trained horses, there were obvious differences in microscopic structure compared with those from the untrained horses. Moderate, industry-standard levels of exercise used to prepare young horses for racing induced the formation of new bone in non-bone spaces in bone tissue, such that the bone organ should better withstand later increased levels of exercise.

Animals↗
PubMed1998

Cytoskeletal regulation of human salivary secretion studied by high resolution electron microscopy and confocal laser microscopy.

To study the cell regulation mechanisms of human salivary secretion, surgical specimens of human parotid and submandibular glands were treated in vitro with isoproterenol (beta-agonist), carbachol (muscarinic agonist), and cytochalasin D (microfilament disruptive agent), and morphological changes occurring in serous acinar cells were observed. Control acinar cells treated without secretagogues exhibited only occasional examples of exocytosis. Microfilaments, revealed by transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM) of F-actin fluorescence stained by rhodamine-phalloidin, were localized underneath the luminal membrane to separate the secretory granules from the luminal membrane. Following isoproterenol treatment, secretory granules made direct contact with the luminal membrane and many omega-shaped exocytotic profiles appeared. TEM and scanning electron microscopy (SEM) showed these profiles to be of granule size or somewhat smaller and to be provided on their cytoplasmic surface with coated pits. Furthermore, CLSM detected the appearance of F-actin fluorescence around the exocytosed granule membranes. Carbachol treatment also evoked the formation in acinar cells of omega-shaped exocytotic profiles some of which were larger than the granules and which exhibited neither coated pits nor associated F-actin fluorescence. To determine if microfilaments regulate the post-exocytotic process of membrane retrieval, we combined isoproterenol treatments with cytochalasin D or carbachol. Following these treatments, F-actin fluorescence surrounding the exocytosed membrane was dispersed or diffused and the exocytotic profiles enlarged remarkably. These results led to the hypothesis that exo/endocytotic processes in human salivary serous acinar cells are regulated differently under autonomic receptor control mediated by microfilaments.

Adult↗
PubMed2004

Contribution of electron and confocal microscopy in the study of Leishmania-macrophage interactions.

Promastigotes of the protozoan parasite genus Leishmania are inoculated into a mammalian host when an infected sand fly takes a bloodmeal. Following their opsonization by complement, promastigotes are phagocytosed by macrophages. There, promastigotes differentiate into amastigotes, the form of the parasite that replicates in the phagolysosomal compartments of host macrophages. Although the mechanisms by which promastigotes survive the microbicidal consequence of phagocytosis remain, for the most part, to be elucidated, evidence indicates that glycoconjugates play a role in this process. One such glycoconjugate is lipophosphoglycan, an abundant promastigote surface glycolipid. Using quantitative electron and confocal laser scanning microscopy approaches, evidence was provided that L. donovani promastigotes inhibit phagolysosome biogenesis in a lipophosphoglycan-dependent manner. This inhibition correlates with an accumulation of periphagosomal F-actin, which may potentially form a physical barrier that prevents L. donovani promastigote-containing phagosomes from interacting with endocytic vacuoles. Inhibition of phagosome maturation may constitute a strategy to provide an environment propitious to the promastigote-to-amastigote differentiation.

Animals↗
PubMed1997

Constitutive expression of ICAM-1 in rat microvascular systems analyzed by laser confocal microscopy.

The present study aimed to demonstrate constitutive expression of the intercellular adhesion molecule (ICAM)-1 among arterioles, capillaries, and venules in the mesentery and liver and to examine the interaction between cultured endothelial cells and leukocytes in rats. ICAM-1 expression in the microvessels in vivo was visually demonstrated by laser confocal fluorescence microscopy. A monoclonal antibody against rat ICAM-1 (1A29) was labeled with fluorescein isothiocyanate, and the binding ratio between the fluorescence and immunoglobulin was determined for data calibration. Intravascularly administered fluorescein isothiocyanate-labeled 1A29 was distributed heterogeneously among the hierarchy of microvessels in the mesentery: postcapillary venules were the major portion expressing ICAM-1 constitutively, and the density of 1A29 bound to their endothelium was at least 10 times higher than that in true capillaries and arterioles in the same mesentery. On the other hand, the liver expressed ICAM-1 abundantly in sinusoids to the extent similar to that in central venules. These results suggest that postcapillary venules serve as an active gateway with the readiness to help adhere circulating leukocytes exposed to proinflammatory stimuli in acute inflammation.

Animals↗
PubMed1998

Gross anatomy of the muscle systems of Fasciola hepatica as visualized by phalloidin-fluorescence and confocal microscopy.

Neuropeptides, biogenic amines and acetylcholine are expressed abundantly within the nervous systems of parasitic flatworms, and are particularly evident in the innervation of the musculature. Such associations have implicated the nervous system in locomotion, host attachment and reproductive co-ordination. Information on the muscle systems of parasitic flatworms is generally sparse, in particular those muscles associated with the reproductive system, intestinal tract and attachment apparatus. Also, the use of sectioned material has left description of the 3-dimensional organization of the musculature largely unrecorded. Using fluorescein isothiocyanate (FITC)-labelled phalloidin as a site-specific probe for filamentous actin, applied to whole-mount preparations of adult Fasciola hepatica and examined by confocal scanning laser microscopy, the present work reports on the organization of the major muscle systems in this trematode parasite. A highly regular array of outer circular, intermediate longitudinal and inner diagonal fibres distinguishes the body wall musculature, which is also involved in the development of both ventral and oral suckers. Circular fibres dominate the duct walls of the male and female reproductive systems, whereas the muscles of the intestinal tract have a somewhat diffuse arrangement of fibres. An understanding of the structural complexity of the muscle systems of parasitic flatworms is considered as fundamental to the interpretation of results from physiological experiments.

Actins↗
PubMed1996

Three-dimensional fluorescence in situ hybridization. Chromosomal studies on nuclei from cytogenetic preparations by confocal microscopy.

OBJECTIVE: To determine the three-dimensional (3D) position of target sequences and chromosomal volumes in interphase human nuclei by confocal laser scanning microscopy (CLSM) by using the heterochromatin part of the long arm of human chromosome Y (HPLAHC) as a target for specific DYZ1 probes, then by D10Z1 probes specific to the centromere of chromosome 10. STUDY DESIGN: Fluorescence in situ hybridization information inside chromosomal preparations was obtained with FITC-labelled probes and propidium iodide (PI) as a DNA-specific stain. To have a control in the experiment, HPLAHC Y was taken as a model of a domain and the centromere of chromosome 10 as a model of a single centromere spot. To have access to their 3D visualization, we selected FITC and PI patterns of fluorescence when optical slices were obtained and used a 3D reconstruction software. RESULTS: Labelling of the target by the probes was characteristic of Y heterochromatin and chromosome 10 centromere localizations and allowed observation of their domain in the x, y and z directions. CONCLUSION: This work was performed on two sets of 30 stained interphase nuclei. Deformations were confirmed by fluorescent spherical beads mounted in the same medium and scanned in the same conditions.

Cell Nucleus↗
PubMed1993

Analysis of volumetric changes in rat pancreatic islets under osmotic stress using laser scanning confocal microscopy.

Analysis of the volumetric changes in rat pancreatic islets undergoing shrinkage and/or swelling due to osmotic stress is essential for understanding the mechanism of mass transport between cells and their environment and for optimizing cryopreservation protocols. Addition and removal of cryoprotective additives is an integral component of all cryopreservation processes. We have used laser scanning confocal microscopy (LSCM) of acridine orange/propidium iodide (AO/PI) stained Islets of Langerhans to analyze the effects of osmotic stress induced by exposure to varying concentrations of the cryoprotectant dimethyl sulfoxide (DMSO), on the islet volume at two temperatures 23 degrees C and 15 degrees C. Experiments were conducted by mounting a single islet onto a unique freeze-thaw-perfusion stage on which the system temperature and the chemical composition of the solutions can be precisely controlled. The bathing medium of the islet was rapidly changed from isotonic saline to the desired DMSO osmolality to produce a defined osmotic stress, and the islet was imaged simultaneously using 488 nm argon laser. Three to seven serial sections were obtained through each islet at increments varying between 15 microns and 20 microns. The three-dimensional (3-D) image was segmented into islet and non-islet regions using a combination of median filtering, gray level thresholding and region labeling, and the islet volume was computed by counting voxels. Further, a special analysis algorithm was applied to identify shape changes both locally and globally throughout the islet volume.

Algorithms↗
PubMed1991

Modulation of hexokinase association with mitochondria analyzed with quantitative three-dimensional confocal microscopy.

Hexokinase isozyme I is proposed to be associated with mitochondria in vivo. Moreover, it has been suggested that this association is modulated in coordination with changes in cell metabolic state. To test these hypotheses, we analyzed the subcellular distribution of hexokinase relative to mitochondria in paraformaldehyde-fixed astrocytes using immunocytochemistry and quantitative three-dimensional confocal microscopy. Analysis of the extent of colocalization between hexokinase and mitochondria revealed that approximately 70% of cellular hexokinase is associated with mitochondria under basal metabolic conditions. In contrast to the immunocytochemical studies, between 15 to 40% of cellular hexokinase was found to be associated with mitochondria after fractionation of astrocyte cultures depending on the exact fractionation conditions. The discrepancy between fractionation studies and those based on imaging of distributions in fixed cells indicates the usefulness of using techniques that can evaluate the distributions of "cytosolic" enzymes in cells whose subcellular ultrastructure is not severely disrupted. To determine if hexokinase distribution is modulated in concert with changes in cell metabolism, the localization of hexokinase with mitochondria was evaluated after inhibition of glucose metabolism with 2-deoxyglucose. After incubation with 2-deoxyglucose there was an approximate 35% decrease in the amount of hexokinase associated with mitochondria. These findings support the hypothesis that hexokinase is bound to mitochondria in rat brain astrocytes in vivo, and that this association is sensitive to cell metabolic state.

Animals↗
PubMed1992

Localization of extracellular matrix components in developing mouse salivary glands by confocal microscopy.

The importance of the extracellular matrix (ECM) in epithelial-mesenchymal interactions in developing organisms is well established. Proteoglycans and interstitial collagens are required for the growth, morphogenesis, and differentiation of epithelial organs and the distribution of these molecules has been described. However, much less is known about other ECM macromolecules in developing epithelial organs. We used confocal microscopy to examine the distribution of laminin, heparan sulfate (BM-1) proteoglycan, fibronectin, and collagen types I, IV, and V, in mouse embryonic salivary glands. Organ rudiments were isolated from gestational day 13 mouse embryos and cultured for 24, 48, or 72 hours. Whole mounts were stained by indirect immunofluorescence and then examined using a Zeiss Laser Scan Microscope. We found that each ECM component examined had a distinct distribution and that the distribution of some molecules varied with culture time. Laminin was mainly restricted to the basement membrane. BM-1 proteoglycan was concentrated in the basement membrane and also formed a fine network throughout the mesenchyme. Type IV collagen was mainly located in the basement membrane of the epithelium, but it was also present throughout the mesenchyme. Type V collagen was distributed throughout the mesenchyme at 24 hours, but at 48 hours was principally located in the basement membrane. Type I collagen was distributed throughout the mesenchyme at all culture times, and accumulated in the clefts and particularly at the epithelial-mesenchymal interface as time in culture increased. Fibronectin was observed throughout the mesenchyme at all times.

Animals↗
PubMed2003

Glutamate triggers rapid glucose transport stimulation in astrocytes as evidenced by real-time confocal microscopy.

Glutamate stimulates glycolysis in astrocytes, a phenomenon that couples astrocytic metabolism with neuronal activity. However, it is not known whether glutamate also affects glucose transporter-1 (GLUT1), the transporter responsible for glucose entry into astrocytes. To address this question, two different real-time single-cell hexose uptake assays were applied to cultured hippocampal astrocytes using confocal epifluorescence microscopy. Glutamate caused a twofold to threefold increase in the zero-trans uptake rates of the fluorescent hexoses 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG) and 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-6-deoxyglucose (6-NBDG). Galactose uptake, determined by the calcein volumetric assay, was stimulated to a similar extent, confirming the fluorescent hexose data, and also demonstrating that glutamate stimulation is a Vmax effect. Remarkably, glucose transport stimulation developed fully inside 10 sec, which is 100 times faster than acute stimulations of glucose transport in other cell types. Glutamate did not significantly affect the rate of 6-NBDG uptake by GLUT1-expressing epithelial Clone 9 cells, suggesting that an astrocyte-specific factor is required for transport stimulation. We conclude that glucose transport stimulation occurs early during astrocytic activation by glutamate, which provides a novel regulatory node to current models of brain energy metabolism. This mechanism should also be considered for the interpretation of functional imaging data based on hexoses.

4-Chloro-7-nitrobenzofurazan↗
PubMed1997

Characterization of pars intermedia connections in amphibians by biocytin tract tracing and immunofluorescence aided by confocal microscopy.

Biocytin, recently introduced in neuroanatomical studies, was used as a retrograde tract tracer in combination with immunofluorescence in order to analyse the neurochemical characters of some central neuronal projections to the pars intermedia in two amphibian species, the anuran Rana esculenta and the urodele Triturus carnifex. After biocytin insertions in the pars intermedia, neurons became retrogradely labelled in the suprachiasmatic hypothalamus and the locus coeruleus of the brainstem in both species. Some scattered biocytin-labelled neurons were observed in the preoptic area. Moreover, working on the same sections, immunofluorescence revealed a number of codistributions and, in some cases, colocalization in the same neurons of biocytin labellings and immunopositivity for (1) tyrosine hydroxylase in the suprachiasmatic hypothalamus and the locus coeruleus of Rana and Triturus, (2) gamma-aminobutyric acid in the suprachiasmatic hypothalamus of Rana and Triturus and (3) neuropeptide Y in the suprachiasmatic hypothalamus of Rana. The specificity of such colocalizations was fully confirmed using dual-channel confocal laser scanning microscopy analysis.

Afferent Pathways↗
PubMed1992

Three-dimensional images of Ramón y Cajal's original preparations, as viewed by confocal microscopy.

The drawings prepared by Santiago Ramón y Cajal have an inherent beauty as well as being accurate and arose partly as a result of the difficulty contemporary photographic methods had in reproducing informative images of the thick sections he used. Modern methods are better and in this brief article Alan Boyde presents some three-dimensional images of these historical preparations, generated by confocal microscopy.

Animals↗
PubMed1995

Combined analysis of in situ hybridization, cell cycle and structural markers using reflectance and immunofluorescence confocal microscopy.

A method for the simultaneous detection of mRNA by reflectance in situ hybridization (RISH), cell cycle and structural markers by immunofluorescence using confocal laser scanning microscopy is presented. The mRNA expression of two ras-related genes rhoB and rhoC was analysed in human breast cancer cell lines and human histological specimens (breast cancer tissues and skin biopsies). In breast cancer cell lines, the conditions were optimized to detect RNA-RNA hybrids and DNA synthesis after pulse-labelling with bromodeoxyuridine. Endonuclease-exonuclease digestion, which allows the accessibility to specific antibodies of halogenated pyrimidine molecules, was carried out following ISH. Finally, cytokeratin or vimentin staining was performed. The detection of signals, arising from 1-nm colloidal gold particles without silver enhancement, by reflectance confocal laser scanning microscopy is described. Bromodeoxybiridine DNA markers and cytokeratin/vimentin staining were detected concomitantly using different fluorochromes. To allow comparative expression of two related genes, the mRNA of rhoB and rhoC were detected using digoxigenin- or biotin-labelled riboprobes and, after 3-D imaging, a detailed analysis by optical horizontal (x, y) and vertical (x, z) sectioning was undertaken. The subsequent bromodeoxyuridine detection procedure permitted to us explore the specific transcription of these two genes during S and non-S phases. This method allows the identification and localization of several subcellular components in cells within a complex tissue structure and makes it possible to analyse further transcript localization in relation to the function of the encoded protein and to the cell cycle.

Biomarkers↗
PubMed1992

Fibronectin and laminin in transverse tubules of cardiac myocytes studied by laser confocal microscopy and immunocytochemistry.

The distribution of two non-collagenous glycoproteins of high molecular weight, fibronectin (FN) and laminin (LMN), was investigated in myocardial cells from the ventricle of rats, and from biopsies collected from the auricle of patients undergoing a coronary bypass operation. In order to elucidate the expression of FN and LMN across cells, non-invasive serial sectioning has been carried out by laser scanning confocal microscopy of frozen, immunostained tissue sections. In addition, immunoelectron microscopy was used to study the distribution of these antigens at higher magnifications. These studies show that FN is part of the basement membrane of the surface sarcolemma of both ventricular and atrial cells, in addition to being an abundant protein of the extracellular matrix (ECM). Along transverse tubular(TT)-membranes, FN was only detected in tubules exceeding 200 nm in diameter. Even here, the intensity of labelling varied greatly and was generally low. By contrast, a heavy investment of LMN was organized in the basal lamina along the surface sarcolemma and along ramifications of the entire TT-system in ventricular heart muscle cells. In this way, the network of TT-membrane systems of working heart muscle cells provides a supply of LMN to all depths of the myocardial fibre. In human atrial muscle cells, a regular TT-system appears to be absent. Instead occasional, deep sarcolemmal invaginations occur with diameters of 300-500 nm, the surfaces of which are also invested with LMN. The significance of the present findings has been discussed, with special reference to LMN as a possible component of a series of proteins involved in transmembrane communication between the ECM and the sarcoplasm.

Animals↗
PubMed1990

Cardiac gap junctions in rat ventricle: localization using site-directed antibodies and laser scanning confocal microscopy.

Antibodies were raised to three synthetic peptides, each constructed to match a different cytoplasmic region of the 43kD protein of the cardiac gap junction. These antibodies have all been used for immunohistochemical localization of gap junctions in the rat ventricle. Each is shown to have different staining characteristics which provide supporting evidence for the model proposed for the configuration of the gap junction protein within the membrane. Laser scanning confocal microscopy has confirmed that the localization of ventricular myocyte gap junctions is solely within intercalated disks. This technique is demonstrated to provide a greater overview of the three dimensional arrangement of the junctions than other microscopical techniques. Larger gap junctions occur typically at the periphery of the disk, with smaller junctions situated in the central zone. The analysis of the number and distribution of gap junctions over large volumes of myocardial tissue (three-dimensional tomography) is now feasible with this approach, and may be used to delineate conduction pathways.

Animals↗
PubMed1994

Vagal innervation of the rat pylorus: an anterograde tracing study using carbocyanine dyes and laser scanning confocal microscopy.

In an attempt to identify the distribution and structure of vagal fibers and terminals in the gastroduodenal junction, vagal efferents were labeled in vivo by multiple injections of the fluorescent carbocyanine dye DiA into the dorsal motor nucleus (dmnX), and vagal afferents were anterogradely labeled by injections of DiI into the nodose ganglia of the same or separate rats. Thick frontal cryostat sections were analysed either with conventional or laser scanning confocal microscopy, using appropriate filter combinations and/or different wavelength laser excitation to distinguish the fluorescent tracers. Vagal efferent terminal-like structures were present in small ganglia within the circular sphincter muscle, which, in the absence of a well-developed, true myenteric plexus at this level, represent the myenteric ganglia. Furthermore, vagal efferent terminals were also present in submucosal ganglia, but were absent from mucosa, Brunner's glands and circular muscle fibers. Vagal afferent fibers and terminal-like structures were more abundant than efferents. The most prominent afferent terminals were profusely branching, large net-like aggregates of varicose fibers running within the connective tissue matrix predominantly parallel to the circular sphincter muscle bundles. Profusely arborizing, highly varicose endings were also present in large myenteric ganglia of the antrum and duodenum, in the modified intramuscular ganglia, and in submucosal ganglia. Additionally, afferent fibers and terminals were present throughout the mucosal lining of the gastroduodenal junction. The branching patterns of some vagal afferents suggested that individual axons produced multiple collaterals in different compartments. NADPH-diaphorase positive, possibly nitroxergic neurons were present in myenteric ganglia of the immediately adjacent antrum and duodenum, and fine varicose fibers entered the sphincter muscle from both sides, delineating the potential vagal inhibitory postganglionic innervation. These morphological results support the view of a rich and differentiated extrinsic neural control of this important gut region as suggested by functional studies.

Animals↗
PubMed1994

Confocal microscopy to analyze cytosolic and nuclear calcium in cultured vascular cells.

With the development of calcium-sensitive fluorescent dyes and videomicroscopic imaging, several investigators have located the changes in intracellular calcium in the cytoplasm, in the perinuclear region, and possibly in the nucleus. However, the presence of calcium in the nucleus is often difficult to ascertain because the fluorescence derived from the perinuclear area interferes with that of the nucleus. We have used confocal microscopy together with two calcium-sensitive dyes [acetoxymethyl esters of fluo 3 (fluo 3-AM) and rhod 2 (rhod 2-AM)] to analyze the cytosolic and nuclear calcium distribution in vascular smooth muscle and endothelial cells studied at rest and after stimulation with receptor-dependent (angiotensin, vasopressin) and receptor-independent (KCl) stimuli. With fluo 3-AM, the baseline fluorescence was located in the cytoplasm but was slightly higher in the nucleus. With all stimuli, the fluorescence intensity increased in both compartments but remained more pronounced within the nucleus. Yet, after calibration, the cytosolic calcium concentration was greater than that of the nucleus at rest and was equally high after stimulation, suggesting different properties of fluo 3 in the cytosol and in the nucleus. With rhod 2-AM, baseline fluorescence was low in the nucleus and high in the cytosol. Cell stimulation caused an initial increase in cytosolic calcium with no change in the nucleus followed by a rise in both compartments. Thus the stimulation of vascular cells is associated with marked increases in cytosolic and nuclear calcium. Fluo 3-AM seems to be a better indicator of nuclear calcium than rhod 2-AM. The increases in nuclear calcium induced by angiotensin II and vasopressin may contribute to their cell proliferative effect.

Aniline Compounds↗