Lucie Kubínová

Topological Properties and Spatial Organization of Villous Capillaries in Normal and Diabetic Placentas

Spatial arrangement and complexity of the capillary bed of placental terminal villi were analyzed in 9 normal and 11 diabetic placentas. Specimens were taken by systematic random sampling, fixed and stained in toto, and embedded in paraffin. Fifteen fields of view were sampled systematically from 120-µm-thick sections of specimens and examined using a confocal laser scanning microscope. Series of thin optical sections of terminal villi and their developmental forms were recorded by the confocal microscope and used as initial data for three-dimensional visualization of the spatial arrangement of villous capillaries. Vascular topology and branching were studied by focusing through the villus, making a schematic drawing of the villous capillary bed and counting redundant capillary connections. It was found that the basic arrangement of villous capillaries is similar in both normal and diabetic placentas. Nevertheless, the proportion of simple forms of the capillary bed without redundant connections is significantly higher in normal placentas and the mean number of redundant connections per villus is significantly higher in diabetic placentas. It is concluded that both the longitudinal growth and branching of capillaries contribute to the increase in the placental capillary bed in late gestation and that the capillary bed of diabetic villi is more complicated due to more intense capillary branching.

Frequency of M-Cadherin-stained Satellite Cells Declines in Human Muscles During Aging

To answer the question of whether the satellite cell pool in human muscle is reduced during aging, we detected satellite cells in 30-μm-thick transverse sections under the confocal microscope by binding of M-cadherin antibody. The basal lamina was detected with laminin. Nuclei were stained with bisbenzimide or propidium iodide. Satellite cells were counted by applying the disector method and unbiased sampling design. To determine if there are age-related differences in muscle fiber types, morphometric characteristics of muscle fibers were examined on thin sections stained for myofibrillar ATPase. Autopsy samples of vastus lateralis muscle from six young (28.7 ± 2.3 years) and six old (70.8 ± 1.3 years) persons who had suffered sudden death were analyzed. Numbers of satellite cells per fiber length (Nsc/Lfib) and number of satellite cells per total number of nuclei (satellite cell nuclei + myonuclei) (Nsc/Nnucl) were significantly lower in the old group (p<0.05). We demonstrate the importance of proper sampling and counting in estimation of sparsely distributed structures such as satellite cells. Our results support the hypothesis that the satellite cell fraction declines during aging.

Flo11p, drug efflux pumps, and the extracellular matrix cooperate to form biofilm yeast colonies

Biofilm yeast colonies are complex structures that form through cooperative action of constituent cells and provide a protective environment for cell growth.

Architecture of developing multicellular yeast colony: spatio-temporal expression of Ato1p ammonium exporter

Yeasts, when growing on solid surfaces, form organized multicellular structures, colonies, in which cells differentiate and thus possess different functions and undergo dissimilar fate. Understanding the principles involved in the formation of these structures requires new approaches that allow the study of individual cells directly in situ without needing to remove them from the microbial community. Here we introduced a new approach to the analysis of whole yeast microcolonies either containing specific proteins labelled by fluorescent proteins or stained with specific dyes, by two-photon excitation confocal microscopy. It revealed that the colonies are covered with a thin protective skin-like surface cell layer which blocks penetration of harmful compounds. The cells forming the layer are tightly connected via cell walls, the presence of which is essential for keeping of protective layer function. Viewing the colonies from different angles allowed us to reconstruct a three-dimensional profile of the cells producing ammonium exporter Ato1p within developing microcolonies growing either as individuals or within a group of microcolonies. We show that neighbouring microcolonies coordinate production of Ato1p-GFP. Ato1p itself appears synchronously in cells, which do not originate from the same ancestor, but occupy specific position within the colony.

Three-dimensional study of the capillary supply of skeletal muscle fibres using confocal microscopy.

Three-dimensional (3D) study of capillary network of individual muscle fibres in rat extensor digitorum longus (EDL) and soleus (SOL) muscles is presented. Stereology and 3D reconstruction techniques were applied to stacks of serial optical sections recorded by a confocal microscope from thick muscle slices. The results suggest that SOL muscle fibres have a larger surface area and volume as well as a larger length of capillaries per fibre length than EDL. On the other hand, these two muscles have a similar ratio of capillary length to fibre surface area. The 3D approach to evaluation of muscle fibre capillarization brings many advantages over traditional measurements made on single muscle sections and could also be applied to the study of angiogenesis in other tissues.

Comparison of Several Digital and Stereological Methods for Estimating Surface Area and Volume of Cells Studied by Confocal Microscopy

BACKGROUND The implementation of different methods for estimating the surface area and volume of cells studied by confocal microscopy was developed. The methods were compared from the point of view of their precision, applicability and efficiency. METHODS Interactive stereological methods (spatial grid method, fakir method, Cavalieri principle) as well as automatic digital methods (digital Crofton method, voxel counting, triangulation method, iso-intensity contouring method) were considered. The methods were tested on model geometrical solids and on real volume images consisting of a stack of serial sections encompassing entire tobacco BY-2 cells or cell chains. RESULTS It is shown that many of the studied methods are very precise when applied to cells of simple or moderately complex shapes. The automatic digital methods are fast and precise but their applicability is limited by the necessity to segment automatically the object surface and to find an optimal resolution. This limitation is not present in stereological methods which are applied interactively and thus are more time-consuming. CONCLUSIONS The presented implementations of the fakir method and the Cavalieri principle enable interactive, unbiased and efficient estimation of the cell surface area and volume. The recommended steps for measuring the surface area and/or volume of objects studied by confocal microscopy are described.

Functional heterogeneity of Thy-1 membrane microdomains in rat basophilic leukemia cells

Antibody‐mediated cross‐linking of Thy‐1 glycoprotein on the surface of rat mast cells and rat basophilic leukemia (RBL) cells initiates biochemical events which culminate in secretion of allergy mediators. Thy‐1, like some other glycosylphosphatidylinositol (GPI)‐anchored proteins, forms detergent‐insoluble complexes containing protein tyrosine kinases (PTK) and some other molecules which are implicated in the signaling pathway. On the surface of a rat mast cell there are more than 106 Thy‐1 molecules; however, it is not known which fraction of them is involved in transmembrane signaling, and what exactly is the heterogeneity of Thy‐1 complexes. Using sucrose density gradient ultracentrifugation of detergent‐lysed RBL cells we found that the density of Thy‐1 complexes depended on the detergent used and the lysis conditions employed. Sepharose 4B gel chromatography fractionation followed by density gradient ultracentrifugation revealed both size and density heterogeneity of Thy‐1 and Lyn PTK complexes. Cross‐linking of surface Thy‐1 caused significant changes in the density of these complexes, and an increase in Lyn kinase activity in low/medium‐density fractions. Thy‐1 in low‐density fractions was relatively resistant to cleavage with phosphatidylinositol‐specific phospholipase C (PI‐PLC). Interestingly, removal of only a small fraction of surface Thy‐1 by PI‐PLC abolished the cell activation as determined by tyrosine phosphorylation of certain proteins. When Triton X‐100 lysates were fractionated at 12 000 × g, about 50 % of Thy‐1 remained associated with the nuclear/cytoskeleton pellet; this fraction of Thy‐1 exhibited an increased sensitivity to PI‐PLC. Confocal laser scanning microscopy on fixed cells revealed that the total Thy‐1 was relatively homogeneously distributed over the plasma membrane, whereas the PI‐PLC‐resistant Thy‐1 was found mostly in small clusters. The combined data suggest that specialized membrane microdomains enriched in Thy‐1 with increased sensitivity to PI‐PLC are directly involved in coupling Thy‐1 aggregation to transmembrane signaling.

The branching pattern of villous capillaries and structural changes of placental terminal villi in type 1 diabetes mellitus

Maternal diabetes is associated with changes of the placental structure. These changes include great variability of vascularity manifested by strikingly hypovascular as well as hypervascular terminal villi. In this paper, normal placental terminal villi and pathological villi of type 1 diabetic placentas were compared concerning the structure of villous stroma, spatial arrangement of villous capillary bed and quantitative assessment of capillary branching pattern. Formalin fixed and paraffin embedded specimens of 14 normal and 17 Type 1 diabetic term placentas were used for picrosirius staining, vimentin and desmin immunohistochemistry and confocal microscopy. 3D models of villi and villous capillaries were constructed from stacks of confocal optical sections. Hypervascular as well as hypovascular villi of diabetic placenta displayed changed structure of villous stroma, i.e. the collagen envelope around capillaries looked thinner and the network of collagen fibers seemed less dense. The desmin immunocytochemistry has shown that stromal cells of hypervascular as well as hypovascular villi appeared nearly or completely void of desmin filaments. In comparison with normal villi, capillaries of hypovascular villi had a smaller diameter and displayed a markedly wavy course whereas in hypervascular villi numerous capillaries occurred in reduced stroma and often had a large diameter. The quantitative assessment of capillary branching has shown that villous capillaries are more branched in diabetic placentas. It is concluded that type 1 maternal diabetes enhances the surface area of the capillary wall by elongation, enlargement of diameter and higher branching of villous capillaries and disrupts the stromal structure of terminal villi.

Spatial arrangement of fetal placental capillaries in terminal villi: a study using confocal microscopy.

Abstract The spatial arrangement of capillaries was studied in terminal villi of placentas at term by using confocal microscopy and methods for different types of 3-D reconstructions. Fixed specimens embedded in paraffin or glycol methacrylate resin were cut and stained with eosin. Digitized images of serial optical sections (approximately 0.5 µm) of individual terminal villi lying completely inside physical sections (100 µm) were captured by confocal laser scanning microscopy and analyzed. Topological reconstruction of the capillary bed and measurements of its Euler number, surface and volume rendering and wire-frame visualization were performed. Our findings showed that villous capillaries are arranged either in a single loop or in a more or less complicated anastomosing system. The results suggest that the combination of confocal microscope capture, methods for 3-D rendering and characterization of topological features reveals valuable information about spatial relationships of tissues in placental villi and the arrangement of the villous microcirculation, e.g. about the branching pattern of capillaries.

Stereological methods based on point counting and unbiased counting frames for two-dimensional measurements in muscles: comparison with manual and image analysis methods

SummaryStereological methods, using the principle of point counting and unbiased counting frames, for the estimation of muscle area, total fibre area, number of muscle fibres and mean fibre area are described in detail. Their practical application is demonstrated on cross-sections of the rat soleus muscles. It is shown that the efficiency of these methods is high and their results are comparable with those achieved by the conventional manual and image analysis methods. The main advantages of two-dimensional stereological methods in muscle morphometry are pointed out: measurements are made directly on specimens under the microscope and in the simplest implementation do not require sophisticated and expensive technical equipment. Furthermore, unbiased results are obtained, no segmentation and edge effect problems arise and the quantity of work invested in stereological estimation is reasonable.Based upon the study of the efficiency of used stereological methods, a suitable test system for muscle morphometry is proposed.