3D analysis of capillary network in skeletal muscle of obese insulin-resistant mice.

In obesity, the skeletal muscle capillary network regresses and the insulin-mediated capillary recruitment is impaired. However, it has been shown that in the early stage of advanced obesity, an increased functional vascular response can partially compensate for other mechanisms of insulin resistance. The present study aimed to investigate the changes in the capillary network around individual muscle fibres during the early stage of obesity and insulin resistance in mice using 3D analysis. Capillaries and muscle fibres of the gluteus maximus muscles of seven high-fat-diet-induced obese and insulin-resistant mice and seven age-matched lean healthy mice were immunofluorescently labelled in thick transverse muscle sections. Stacks of images were acquired using confocal microscope. Capillary network characteristics were estimated by methods of quantitative image analysis. Muscle fibre typing was performed by histochemical analysis of myosin heavy chain isoforms on thin serial sections of skeletal muscle. Capillary length per muscle fibre length and capillary length per muscle fibre surface were increased by 27% and 23%, respectively, around small muscle fibres in obese mice, while there were no significant comparative differences around large fibres of obese and lean mice. Furthermore, the capillarization was larger around small compared to large fibres and there was a shift toward fast type myosin heavy chain isoforms, with no significant changes in muscle fibre diameters, tortuosity and anisotropy in obese mice. Overall, the results show that obese insulin-resistant mice have selective increase in capillarization around small predominantly intermediate muscle fibres, which is most likely related to the impaired glucose metabolism characteristic of type 2 diabetes.

Confocal stereology: an efficient tool for measurement of microscopic structures.

Quantitative measurements of geometric forms or counting of objects in microscopic specimens is an essential tool in studies of microstructure. Confocal stereology represents a contemporary approach to the evaluation of microscopic structures by using a combination of stereological methods and confocal microscopy. 3-D images acquired by confocal microscopy can be used for the estimation of geometrical characteristics of microscopic structures by stereological methods, based on the evaluation of optical sections within a thick slice and using computer-generated virtual test probes. Such methods can be used for estimating volume, number, surface area and length using relevant spatial probes, which are generated by specific software. The interactions of the probes with the structure under study are interactively evaluated. An overview of the methods of confocal stereology developed during the past 30 years is presented. Their advantages and pitfalls in comparison with other methods for measurement of geometrical characteristics of microscopic structures are discussed.

Unbiased estimation of chloroplast number in mesophyll cells: advantage of a genuine three-dimensional approach.

Chloroplast number per cell is a frequently examined quantitative anatomical parameter, often estimated by counting chloroplast profiles in two-dimensional (2D) sections of mesophyll cells. However, a mesophyll cell is a three-dimensional (3D) structure and this has to be taken into account when quantifying its internal structure. We compared 2D and 3D approaches to chloroplast counting from different points of view: (i) in practical measurements of mesophyll cells of Norway spruce needles, (ii) in a 3D model of a mesophyll cell with chloroplasts, and (iii) using a theoretical analysis. We applied, for the first time, the stereological method of an optical disector based on counting chloroplasts in stacks of spruce needle optical cross-sections acquired by confocal laser-scanning microscopy. This estimate was compared with counting chloroplast profiles in 2D sections from the same stacks of sections. Comparing practical measurements of mesophyll cells, calculations performed in a 3D model of a cell with chloroplasts as well as a theoretical analysis showed that the 2D approach yielded biased results, while the underestimation could be up to 10-fold. We proved that the frequently used method for counting chloroplasts in a mesophyll cell by counting their profiles in 2D sections did not give correct results. We concluded that the present disector method can be efficiently used for unbiased estimation of chloroplast number per mesophyll cell. This should be the method of choice, especially in coniferous needles and leaves with mesophyll cells with lignified cell walls where maceration methods are difficult or impossible to use.

Blood capillary length estimation from three-dimensional microscopic data by image analysis and stereology.

Studies of the capillary bed characterized by its length or length density are relevant in many biomedical studies. A reliable assessment of capillary length from two-dimensional (2D), thin histological sections is a rather difficult task as it requires physical cutting of such sections in randomized directions. This is often technically demanding, inefficient, or outright impossible. However, if 3D image data of the microscopic structure under investigation are available, methods of length estimation that do not require randomized physical cutting of sections may be applied. Two different rat brain regions were optically sliced by confocal microscopy and resulting 3D images processed by three types of capillary length estimation methods: (1) stereological methods based on a computer generation of isotropic uniform random virtual test probes in 3D, either in the form of spatial grids of virtual "slicer" planes or spherical probes; (2) automatic method employing a digital version of the Crofton relations using the Euler characteristic of planar sections of the binary image; and (3) interactive "tracer" method for length measurement based on a manual delineation in 3D of the axes of capillary segments. The presented methods were compared in terms of their practical applicability, efficiency, and precision.

Ecology of testate amoebae in the Komorany ponds in the Vltava Basin.

Testate amoeba (TA) assemblages were collected in 2005 from four ponds in Komorany (Prague, Czech Republic). An analysis of seasonal taxonomic variability of TA populations and its correlation with the limnological characteristics of the area (temperature, pH, total organic carbon, nitrogen, phosphorus, heavy metals, etc.) was performed. The predominant genera were Difflugia, Arcella, and Centropyxis. The most significant changes in the TA community occurred between March and July. Arcella genus dominated in March and April; in May, Arcella and Centropyxis genera were present in the same amount; in June, Arcella genus disappeared, and Difflugia genus started to dominate the community. A multivariate redundancy analysis showed statistically significant correlations between the environmental parameters and the composition of the TA community. The results indicate a negative correlation between TA quantities and Ni, Cd, PAH, Mn, As, and Pb. TA were also affected by concentrations of NH4(+), NO3(-), and P, as well as by temperature variations. The observed correlations between the species composition and environmental parameters can be used in paleoecological interpretations of fossil TA communities. Our results also prove the suitability of TA as water quality indicators in urban areas.

Visualization of Reinke's crystals in normal and cryptorchid testis.

Within the human testis, Reinke's crystals are found in Leydig cells but their nature and function are poorly understood. The aim of our study was to investigate the properties of Reinke's crystals in man with the normal morphology of the testis (control group) and infertile patients diagnosed with cryptorchidism. 20 biopsies from infertile patients and six biopsies from men with regular spermatogenesis (20-30 years.) were used. Sections of the testis tissue were stained with haematoxylin and eosin and a modified Masson's method. Specimens were observed by bright field, confocal and transmission electron microscopy (TEM). The number of Reinke's crystals in investigated groups was determined applying stereological methods. In both groups, Reinke's crystals were noted within the cytoplasm and nuclei of Leydig cells. Some "free" crystals were found within the interstitial space, outside Leydig cells. Confocal microscopy proved to be very useful in the assessment of the shape and 3D reconstruction of the crystal. TEM analysis confirmed a hexagonal form of the crystal, while crystallographic data on sections of 70-300 nm thickness provided a better insight into the organization of the crystal lattice. Stereological analysis revealed a significant increase in the number of crystals in cryptorchid testes when compared with controls. Increased number of crystals in cryptorchid specimens leads to the assumption that the prolonged exposure to higher (abdominal) temperature might stimulate enzymes involved in the synthesis of the proteins of the crystal. However, the exact molecular nature of the crystal lattice remains in both normal and cryptorchid testis obscure.

A novel method for evaluation of capillarity in human skeletal muscles from confocal 3D images.

A well developed capillary bed is essential for proper function of skeletal muscles. We present for the first time a triple immunofluorescent method suitable for staining capillaries and muscle fibre outlines in thick sections of human skeletal muscle, applying antibodies against collagen IV (in red) and F8 (in green) as well as Ulex europaeus lectin, visualized in green fluorescence. Further, we present possibilities for quantitative evaluation of the capillary network which implies the length of capillaries per unit volume of muscle tissue (Lcap/Vmuscle) and the length of capillaries supplying individual muscle fibres per unit fibre length (Lcap/Lfib), per surface area (Lcap/Sfib) and per volume (Lcap/Vfib) as well as the course of capillaries in the muscle. The latter can be described by the tortuosity, orientation and mean capillary length. To get reasonable results we met the following requirements: i) high quality thick tissue sections, from which 3D image data were acquired; ii) immunofluorescent methods suitable for confocal microscopy; iii) penetration of the fluorescent dyes throughout the tissue section; iv) proper 3D image analysis methods for performing reliable measurements and v) control over relevant tissue deformations. The developed methodology is illustrated by results obtained from autopsy or biopsy samples of three human muscles, i.e. vastus lateralis, multifidus and masseter muscle that exhibit differences in genetic background, innervation, tasks and functional activity.

Nonrigid registration of CLSM images of physical sections with discontinuous deformations.

When biological specimens are cut into physical sections for three-dimensional (3D) imaging by confocal laser scanning microscopy, the slices may get distorted or ruptured. For subsequent 3D reconstruction, images from different physical sections need to be spatially aligned by optimization of a function composed of a data fidelity term evaluating similarity between the reference and target images, and a regularization term enforcing transformation smoothness. A regularization term evaluating the total variation (TV), which enables the registration algorithm to account for discontinuities in slice deformation (ruptures), while enforcing smoothness on continuously deformed regions, was proposed previously. The function with TV regularization was optimized using a graph-cut (GC) based iterative solution. However, GC may generate visible registration artifacts, which impair the 3D reconstruction. We present an alternative, multilabel TV optimization algorithm, which in the examined samples prevents the artifacts produced by GC. The algorithm is slower than GC but can be sped up several times when implemented in a multiprocessor computing environment. For image pairs with uneven brightness distribution, we introduce a reformulation of the TV-based registration, in which intensity-based data terms are replaced by comparison of salient features in the reference and target images quantified by local image entropies.

Local Immune Changes in Early Stages of Inflammation and Carcinogenesis Correlate with the Collagen Scaffold Changes of the Colon Mucosa.

Continuous activation of the immune system inside a tissue can lead to remodelling of the tissue structure and creation of a specific microenvironment, such as during the tumour development. Chronic inflammation is a central player in stimulating changes that alter the tissue stroma and can lead to fibrotic evolution. In the colon mucosa, regulatory mechanisms, including TGF-ß1, avoid damaging inflammation in front of the continuous challenge by the intestinal microbiome. Inducing either DSS colitis or AOM colorectal carcinogenesis in AVN-Wistar rats, we evaluated at one month after the end of each treatment whether immunological changes and remodelling of the collagen scaffold were already in development. At this time point, we found in both models a general downregulation of pro-inflammatory cytokines and even of TGF-ß1, but not of IL-6. Moreover, we demonstrated by multi-photon microscopy the simultaneously presence of pro-fibrotic remodelling of the collagen scaffold, with measurable changes in comparison to the control mucosa. The scaffold was significantly modified depending on the type of induced stimulation. These results suggest that at one month after the end of the DSS or AOM inductions, a smouldering inflammation is present in both induced conditions, since the pro-inflammatory cytokines still exceed, in proportion, the local homeostatic regulation of which TGF-ß1 is a part (inflammatory threshold). Such an inflammation appears sufficient to sustain remodelling of the collagen scaffold that may be taken as a possible pathological marker for revealing pre-neoplastic inflammation.

The estimation error of skeletal muscle capillary supply is significantly reduced by 3D method.

Capillary supply of individual skeletal muscle fibers is usually evaluated from two-dimensional (2D) images of thin transverse sections by the number of capillary profiles around a fiber (CAF). This method is inherently inaccurate and the resulting capillary length measurement errors can be avoided by using an alternative three-dimensional (3D) approach where the mean length of capillaries around individual muscle fibers per fiber length (Lcap/Lfib) is measured from 3D images acquired by confocal microscopy. We quantified the error of the 2D method and its reduction by using a 3D approach in realistic geometrical models of muscle fiber capillary bed and in true muscle samples. In models we showed that Lcap/Lfib was sensitive to different arrangements of capillaries, while CAF underestimated capillarization since it could not detect the increased length of capillary bed. In true muscle samples, we detected statistically significant differences in the capillary supply of control and denervated rat soleus muscles by both 2D and 3D methods. Lcap/Lfib was larger than CAF in control muscles reflecting their more complicated capillary bed. Thus, 3D approach is more sensitive in agreement with the analysis of geometrical models. We conclude that the 3D method, though technically more demanding than 2D method, represents a more precise approach to evaluation of muscle capillarization. Moreover, the 3D method could be applied to other organs and we suggest potential medical applications.

Testate amoebae examined by confocal and two-photon microscopy: implications for taxonomy and ecophysiology.

Testate amoebae (TA) are a group of free-living protozoa, important in ecology and paleoecology. Testate amoebae taxonomy is mainly based on the morphological features of the shell, as examined by means of light microscopy or (environmental) scanning electron microscopy (SEM/ESEM). We explored the potential applications of confocal laser scanning microscopy (CLSM), two photon excitation microscopy (TPEM), phase contrast, differential interference contrast (DIC Nomarski), and polarization microscopy to visualize TA shells and inner structures of living cells, which is not possible by SEM or environmental SEM. Images captured by CLSM and TPEM were utilized to create three-dimensional (3D) visualizations and to evaluate biovolume inside the shell by stereological methods, to assess the function of TA in ecosystems. This approach broadens the understanding of TA cell and shell morphology, and inner structures including organelles and endosymbionts, with potential implications in taxonomy and ecophysiology.

Microtubule Cytoskeleton Remodeling by Nanosecond Pulsed Electric Fields.

Remodeling of nanoscopic structures is not just crucial for cell biology, but it is also at the core of bioinspired materials. While the microtubule cytoskeleton in cells undergoes fast adaptation, adaptive materials still face this remodeling challenge. Moreover, the guided reorganization of the microtubule network and the correction of its abnormalities is still a major aim. This work reports new findings for externally triggered microtubule network remodeling by nanosecond electropulses (nsEPs). At first, a wide range of nsEP parameters, applied in a low conductivity buffer, is explored to find out the minimal nsEP dosage needed to disturb microtubules in various cell types. The time course of apoptosis and microtubule recovery in the culture medium is thereafter assessed. Application of nsEPs to cells in culture media result in modulation of microtubule binding properties to end-binding (EB1) protein, quantified by newly developed image processing techniques. The microtubules in nsEP-treated cells in the culture medium have longer EB1 comets but their density is lower than that of the control. The nsEP treatment represents a strategy for microtubule remodeling-based nano-biotechnological applications, such as engineering of self-healing materials, and as a manipulation tool for the evaluation of microtubule remodeling mechanisms during various biological processes in health and disease.

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.

3D visualization and measurement of capillaries supplying metabolically different fiber types in the rat extensor digitorum longus muscle during denervation and reinnervation.

The aim of this study was to determine whether capillarity in the denervated and reinnervated rat extensor digitorum longus muscle (EDL) is scaled by muscle fiber oxidative potential. We visualized capillaries adjacent to a metabolically defined fiber type and estimated capillarity of fibers with very high oxidative potential (O) vs fibers with very low oxidative potential (G). Capillaries and muscle fiber types were shown by a combined triple immunofluorescent technique and the histochemical method for NADH-tetrazolium reductase. Stacks of images were captured by a confocal microscope. Applying the Ellipse program, fibers were outlined, and the diameter, perimeter, cross-sectional area, length, surface area, and volume within the stack were calculated for both fiber types. Using the Tracer plug-in module, capillaries were traced within the three-dimensional (3D) volume, the length of capillaries adjacent to individual muscle fibers was measured, and the capillary length per fiber length (Lcap/Lfib), surface area (Lcap/Sfib), and volume (Lcap/Vfib) were calculated. Furthermore, capillaries and fibers of both types were visualized in 3D. In all experimental groups, O and G fibers significantly differed in girth, Lcap/Sfib, and Lcap/Vfib, but not in Lcap/Lfib. We conclude that capillarity in the EDL is scaled by muscle fiber size and not by muscle fiber oxidative potential.

Reversible and Irreversible Modulation of Tubulin Self-Assembly by Intense Nanosecond Pulsed Electric Fields.

Tubulin self-assembly into microtubules is a fascinating natural phenomenon. Its importance is not just crucial for functional and structural biological processes, but it also serves as an inspiration for synthetic nanomaterial innovations. The modulation of the tubulin self-assembly process without introducing additional chemical inhibitors/promoters or stabilizers has remained an elusive process. This work reports a versatile and vigorous strategy for controlling tubulin self-assembly by nanosecond electropulses (nsEPs). The polymerization assessed by turbidimetry is dependent on nsEPs dosage. The kinetics of microtubules formation is tightly linked to the nsEPs effects on structural properties of tubulin, and tubulin-solvent interface, assessed by autofluorescence, and the zeta potential. Moreover, the overall size of tubulin assessed by dynamic light scattering is affected as well. Additionally, atomic force microscopy imaging reveals the formation of different assemblies reflecting applied nsEPs. It is suggested that changes in C-terminal modification states alter tubulin polymerization-competent conformations. Although the assembled tubulin preserve their integral structure, they might exhibit a broad range of new properties important for their functions. Thus, these transient conformation changes of tubulin and their collective properties can result in new applications.

Capillary Network Morphometry of Pig Soleus Muscle Significantly Changes in 24 Hours After Death.

Capillary network characteristics are invaluable for diagnostics of muscle diseases. Biopsy material is limited in size and mostly not accessible for intensive research. Therefore, especially in human tissue, studies are performed on autopsy material. To approach the problem whether it is reliable to deduce hypotheses from autopsy material to explain physiological and pathological processes, we studied capillarity in pig soleus muscle 1 and 24 hr after death. Capillaries and muscle fibers were immunofluorescently marked, and images were acquired with a confocal microscope. Characteristics of the capillary network were estimated by image analysis methods using several plugins of the Ellipse program. Twenty-four hours after death, the measured characteristics of the capillary network differ by up to 50% when compared with samples excised 1 hr after death. Muscle fiber diameter, the measured capillary length, and tortuosity were reduced, and capillary network became more anisotropic. The main postmortem change that affects capillaries is evidently geometric deformation of muscle tissue. In conclusion, when comparing results from biopsy samples with those from autopsy samples, the effect of postmortem changes on the measured parameters must be carefully considered.

Methods for compensation of the light attenuation with depth of images captured by a confocal microscope.

A confocal laser scanning microscope (CLSM) enables us to capture images from a biological specimen in different depths and obtain a series of precisely registered fluorescent images. However, images captured from deep layers of the specimen may be darker than images from the topmost layers because of light loss distortions. This effect causes difficulties in subsequent analysis of biological objects. We propose a solution using two approaches: either an online method working already during image acquisition or an offline method assisting as a postprocessing step. In the online method, the gain value of a photomultiplier tube of a CLSM is controlled according to the difference of mean image intensities between the reference and currently acquired image. The offline method consists of two stages. In the first stage, a standard histogram maintaining relative frequencies of gray levels and improving brightness and contrast is created from all images in the series. In the second stage, individual image histograms are warped according to this standard histogram. The methods were tested on real confocal image data captured from human placenta and rat skeletal muscle specimens. It was shown that both approaches diminish the light attenuation in images captured from deep layers of the specimen.

Visualisation of human dental pulp vasculature by immunohistochemical and immunofluorescent detection of CD34: a comparative study.

CD34 is considered a pan-endothelial cell marker for paraffin-embedded sections. In this study, both immunohistochemistry and immunofluorescence were applied in human dental pulp specimens of moderate thickness (10 microm) in order to observe the vasculature of this tissue using CD34. Both techniques revealed a homogenous staining pattern with capillaries and larger vessels showing complete and strong membrane staining reflecting the high capacity of the pulp for regeneration and response to different stimuli. A novel approach in the identification of the pulpal vasculature by Cy5-conjugated anti-CD34 is introduced in this study. By this technique the dense capillary plexus of the sub-odontoblastic region, which is responsible for the reaction of the tissue to any physical or chemical stimuli or pathological condition, can be clearly identified, while immunohistochemistry did not reveal such a detailed staining pattern.

Image-Based Modeling of Blood Flow and Oxygen Transfer in Feto-Placental Capillaries.

During pregnancy, oxygen diffuses from maternal to fetal blood through villous trees in the placenta. In this paper, we simulate blood flow and oxygen transfer in feto-placental capillaries by converting three-dimensional representations of villous and capillary surfaces, reconstructed from confocal laser scanning microscopy, to finite-element meshes, and calculating values of vascular flow resistance and total oxygen transfer. The relationship between the total oxygen transfer rate and the pressure drop through the capillary is shown to be captured across a wide range of pressure drops by physical scaling laws and an upper bound on the oxygen transfer rate. A regression equation is introduced that can be used to estimate the oxygen transfer in a capillary using the vascular resistance. Two techniques for quantifying the effects of statistical variability, experimental uncertainty and pathological placental structure on the calculated properties are then introduced. First, scaling arguments are used to quantify the sensitivity of the model to uncertainties in the geometry and the parameters. Second, the effects of localized dilations in fetal capillaries are investigated using an idealized axisymmetric model, to quantify the possible effect of pathological placental structure on oxygen transfer. The model predicts how, for a fixed pressure drop through a capillary, oxygen transfer is maximized by an optimal width of the dilation. The results could explain the prevalence of fetal hypoxia in cases of delayed villous maturation, a pathology characterized by a lack of the vasculo-syncytial membranes often seen in conjunction with localized capillary dilations.

Three-dimensional reconstructions from non-deparaffinized tissue sections.

Three-dimensional (3-D) reconstruction from microscopic images represents a useful tool for the study of biological structures in embryology and developmental biology. However, it is usually necessary to cope with many difficulties connected with the preparation of specimens. In order to minimize mutual displacement of structures in successive sections, the applicability of non-deparaffinized tissue sections for 3-D reconstruction was tested. Chicken embryos were fixed and stained in toto with eosin and then embedded in paraffin. About 30-mum-thick non-deparaffinized serial sections were used for obtaining initial data for 3-D reconstruction of larger stacks of embryonic bodies using either fluorescence or confocal microscope. The same sections served for both collecting optical serial sections of mesonephros as source images for its 3-D reconstruction, and immunohistochemical detection of fibronectin, laminin and vimentin. It was found that sections with retained paraffin preserve the mutual spatial relationships of tissue components as well as provide an excellent differentiation of structure. It makes the process of 3-D reconstruction easier. The localization of the products of immunohistochemical reactions demonstrated the co-localization of fibronectin and laminin in basal laminas and the presence of vimentin in glomeruli and mesenchymal tissue. The use of non-deparaffinized sections represents a less time consuming and more effective alternative to thin histological sections for the purpose of 3-D reconstruction, and enables further application of material.