Confocal Laser Microscopy Images Research Articles

Differentiation of the body wall musculature in Macrostomum hystricinum marinum and Hoploplana inquilina (Plathelminthes), as models for muscle development in lower Spiralia

Recent studies on the differentiation of the body wall musculature in a medicinal leech and in the free-living plathelminth Macrostomum hystricinum marinum, Beklemischev 1950 provide the first evidence of a complex developmental signalling pattern, possibly involving stem cells and the nervous system, in the organization of the muscle grid formed by developing myocytes. To enhance further our understanding of the ontogenetic and phylogenetic origin of such muscle grids, which consist of circular, longitudinal and diagonal muscle fibres, we have undertaken a study of muscle development in the polyclad flatworm Hoploplana inquilina Wheeler 1894 in collaboration with the Marine Biological Laboratory, Woods Hole. We have also continued our examination of the development of the body wall musculature in M. hystricinum. Both species were studied using rhodamine-phalloidin staining and transmission electron microscopy. Additional visualization of the fluorescent whole mount preparations was performed with confocal laser microscopy and digital image processing. The results of our investigation suggest that: (1) the mechanism of muscle development in H. inquilina supports the deeply rooted concept of bilateral symmetry (right and left longitudinal founder muscle), and (2) a first circular muscle in this species develops on the border between an anterior body unit and the main body; a caudalmost region is less obvious. The presence of a spiral muscle functioning as a circular muscle system of the "head region" points to a separate developmental mechanism for this region and the trunk. In contrast to H. inquilina, where the larval stage forces an intermediate restructuring of the musculature of the body wall before the adult body shape is finally developed, the formation of the body wall musculature of M. hystricinum already seems constrained by the adult body shape.

A flowcell for the study of plaque removal and regrowth

A flowcell has been designed for microscopy of oral biofilm communities (plaque) during their colonization, removal, and subsequent regrowth. These processes were documented using low-magnification darkfield microscopy, high-magnification oil-immersion phase-contrast microscopy, and Confocal Laser Microscopy (CLM). The number of cells and microaggregates (both hereafter referred to as particles) that attached to the flowcell surface from whole saliva, as well as the area covered by these particles, increased linearly with time over a three-hour period. The smallest particle-size-class (1−6 μm 2) contained the greatest number of particles: at least 50% of the total number of particles. The biofilm can be removed using an internal scraping device. Two cycles of cleaning/ regrowth were documented. Cleaning removed 50% of the initial biofilm; regrowth resulted in a four-fold increase in coverage over that initially present. The second cleaning removed 75% of this heavier colonization, and the second regrowth period resulted in a three-fold increase in coverage over that present in the first regrowth. High-resolution photomicrographs and CLM images were obtained that permit morphological analysis of the community structure within the mixed species biofilm in three dimensions. Exclusion-staining (cells are detected by the exclusion of fluorogenic dye) using fluorescein revealed differences in cellular chemistry between cells within a single morphological type.

Cytokine receptor alterations during HIV infection in the human central nervous system

Cytokines are potent factors mediating interactions between the immune and nervous systems. Cytokines released by macrophages/microglia, the predominant immune cell within the brain, have been proposed to modulate neuronal survival and death. In human immunodeficiency virus-encephalitis (HIVE), cytokines could modulate neurologic damage if nervous system cells possessed appropriate receptors. We hypothesized that the populations of neurons vulnerable to the toxic effects of cytokines in HIVE might contain specific receptors for these molecules. We examined the distribution of cytokine receptors in the human brain utilizing fluorescent-labeled cytokines combined with confocal laser microscopy imaging. Phycoerthrin-conjugated interleukin-1β and phycoerythrin-Avidin/biotin conjugated transforming growth factor β1 labeled dendritic processes of neurons in the neocortex. Labeling was abolished by pre-incubation with unlabeled with cytokines. In cases with moderate HIVE, an average 35% increase in intensity of labeling was observed compared to cases without HIVE or with cases with severe HIVE. The patterns of interleukin 2 labeling were not altered in HIVE. These results suggest that neurons susceptible to cytokine-mediated damage during the progression of HIVE display abnormal patterns of cytokine receptor labeling.

The alpha 6 and beta 4 integrin subunits are expressed by smooth muscle cells of human small vessels: a new localization in mesenchymal cells.

The alpha 6 beta 4 integrin complex is generally thought to be expressed by epithelial cells, where it is localized in specific adhesion structures, the hemidesmosomes. Recent observations have suggested a new localization of the beta 4 integrin chain in small vessels, possibly in endothelial cells, i.e., in cells of mesenchymal origin. In the present study we show that (a) the alpha 6 and beta 4 integrin chains are not expressed by endothelial cells, since they are not localized in von Willebrand factor-producing cells; (b) instead, smooth muscle cells of small vessels are intensely positive to antibodies to both alpha 6 and beta 4 intergrin chains; and (c) in some restricted regions of these smooth muscle cells there is a clear colocalization between alpha-smooth muscle actin and alpha 6 and beta 4 integrin chains, suggesting that a new type of cytoskeletal linkage for the alpha 6 beta 4 integrin complex may occur in mesenchyme-derived cells. Our observations are supported by confocal laser microscopy (CLSM) images of specimens labeled by double immunofluorescence. This technical choice was made to take advantage of the higher resolution offered by CLSM in comparison with conventional immunofluorescence. A careful selection of barrier filters was necessary to separate accurately emission and excitation spectra of the fluorochromes used in this study, resulting in an efficient colocalization analysis.

The spatial distribution and relative abundance of gap-junctional connexin40 and connexin43 correlate to functional properties of components of the cardiac atrioventricular conduction system.

Electrical coupling between heart muscle cells is mediated by specialised regions of sarcolemmal interaction termed gap junctions. In previous work, we have demonstrated that connexin42, a recently identified gap-junctional protein, is present in the specialised conduction tissues of the avian heart. In the present study, the spatial distribution of the mammalian homologue of this protein, connexin40, was examined using immunofluorescence, confocal scanning laser microscopy and quantitative digital image analysis in order to determine whether a parallel distribution occurs in rat. Connexin40 was detected by immunofluorescence in all main components of the atrioventricular conduction system including the atrioventricular node, atrioventricular bundle, and Purkinje fibres. Quantitation revealed that levels of connexin40 immunofluorescence increased along the axis of atrioventricular conduction, rising over 10-fold between atrioventricular node and atrioventricular bundle and a further 10-fold between atrioventricular bundle and Purkinje fibres. Connexin40 and connexin43, the principal gap-junctional protein of the mammalian heart, were co-localised within atrioventricular nodal tissues and Purkinje fibres. By applying a novel photobleach/double-labelling protocol, it was demonstrated that connexin40 and connexin43 are co-localised in precisely the same Purkinje fibre myocytes. A model, integrating data on the spatial distribution and relative abundance of connexin40 and connexin43 in the heart, proposes how myocyte-type-specific patterns of connexin isform expression account for the electrical continuity of cardiac atrioventricular conduction.

Confocal Microscopy and Three-Dimensional Image Analysis Methods for Recognition and Fluorometric Measurements: An Application to Inhibitory Glycine Receptor.

Confocal scanning laser microscopy (CSLM) and 3D image analysis have been used for recognition and fluorometric measurements of fluorescent spots in specimens. The methods have been applied to clusters of chloride-dependent glycine receptors on the surface membranes of neurons in the central nervous system. The receptors were labeled with a specific monoclonal antibody and by indirect immunofluorescence. These glycine-activated chloride channels form aggregates that appear as spots with high fluorescence intensity and more or less regular contour, each corresponding to a postsynaptic density. A CSLM recorded the fluorescence within the specimens and described it with digital volumes. Digital 3D image analysis was used for recognition of spots in these volumes and for measurements of their geometric and photometric properties. When these methods were applied to neurons from the goldfish reticulata, the results indicated that the concentration of receptor is relatively stable from one cluster to another but their size and the total number of receptive molecules vary from one synaptic contact to the next. Results of this kind are important in modeling the integrative properties of central neurons.

Cellular mechanism of the insulin-like effect of growth hormone in adipocytes. Rapid translocation of the HepG2-type and adipocyte/muscle glucose transporters.

The cellular mechanism whereby growth hormone (GH) acutely stimulates adipocyte glucose uptake was studied in cultures of primary rat adipocytes differentiated in vitro. Preadipocytes were isolated by collagenase digestion of inguinal fat-pads from young rats and were differentiated in the presence of 3-isobutyl-1-methylxanthine, insulin and dexamethasone. The development of an adipocyte morphology (i.e. lipid inclusions) was observed over 6 days after initiation of differentiation. Coincident with this phenotypic change was an increase in glyceraldehyde-3-phosphate dehydrogenase (GPDH) activity and in cellular content of the HepG2-type (Glut1) and adipocyte/muscle (Glut4) glucose transporter isoforms as determined by Western immunoblotting of total cellular protein. Age-matched undifferentiated cells expressed the Glut1 transporter and low levels of GPDH, but neither accumulated lipid nor exhibited measurable expression of the Glut4 protein. On day 6 after the initiation of differentiation, GH and insulin stimulated 2-deoxy[14C]glucose uptake in a dose- and time-dependent fashion in adipocytes cultured under serum-free conditions for at least 15 h. Western-blot analysis of subcellular fractions revealed that both GH and insulin rapidly (within 20 min) stimulated translocation of the Glut1 and Glut4 proteins from a low-density microsomal fraction to the plasma membrane. Confirmatory evidence was provided in immunocytochemical experiments utilizing antisera directed against the C-terminal region of the Glut4 protein and a fluorescein isothiocyanate-labelled second antibody. Observation of the cells via confocal laser microscopic imaging was consistent with glucose transporter redistribution from an intracellular region to the plasma membrane after treatment with GH or insulin. On the basis of these data, we suggest that the insulin-like effect of GH on adipocyte glucose transport involves translocation of the Glut1 and Glut4 proteins to the plasma membrane. Furthermore, stimulation of glucose-transporter translocation by both GH and insulin may indicate a common cell signalling element between the adipocyte GH and insulin receptors or, alternatively, the existence of multiple cellular mechanisms for stimulating glucose-transporter translocation.

Three-dimensional specimen recording and interactive display using confocal laser microscopy and digital image processing

In this paper we first give a brief overview of three-dimensional specimen recording and display using confocal microscopy and digital image processing. We then concentrate on some practical aspects of using this technique: We investigate how the specimen refractive index and detector integration time will influence image resolution and describe an interactive user interface to the computer environment.