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Virtual reality is increasingly used in medicine for diagnostics, for visualisation of complex structures and for preoperative planning. In interventional radiology, minimally invasive approach could be described with a target point representing the desired needle tip position and an array of all possible trajectories leading to it resembling irregular “cone” or “pyramid”. We present a pilot study of planning a minimally invasive posterior infrazygomatic and suprazygomatic approaches into the pterygopalatine fossa using a solid angle as a measure of size of the approach in five virtually reconstructed heads. The minimally invasive approaches were planned by manually drawing the edges of “pyramids” that described each approach in 3D using virtual reality program Tracer. For each head, a transverse diameter was measured and for each approach a solid angle size, average edge length and estimated area on the skin from where the target point could be reached were calculated. We found that, the solid angle of posterior infrazygomatic approach was significantly larger than suprazygomatic approach (p0.001). Furthermore, the transverse head diameter and solid angle in posterior infrazygomatic approach were negatively correlated (ρ=-0.55, p=0.0002), while transverse head diameter and the estimated area on the skin from where the target point could be reached in the suprazygomatic approach were positively correlated (ρ=0.37, p=0.0206). In conclusion, our findings provide important preliminary evidence on the feasibility of evaluating and comparing different minimally invasive approaches using virtual reality systems, and affirm the validity of solid angle as a measure of the size of the approach.

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.

The main objective of this study was to find out whether the selected chloroplast characteristics measured in the mesophyll layer nearest to the needle surface (i.e., the first mesophyll layer) could be representative for the whole needle cross section. Two chloroplast sampling approaches were applied on Norway spruce needles during the investigation of the effects of different levels of air CO2 concentration and irradiance: (i) sampling only from the first mesophyll layer, and (ii) systematic uniform random (SUR) sampling. The selected characteristics were: (i) chloroplast area, (ii) starch grain area, and (iii) starch areal density on median chloroplast cross sections, and (iv) chloroplast number per unit of needle volume. It was shown that the first mesophyll layer was not representative for estimating all evaluated characteristics except the chloroplast area. Sampling only there caused obtaining slightly biased results, while SUR sampling gave unbiased estimations at the cost of longer measuring time. The major effect of studied factors was in starch areal density and starch grain area, which were larger in sun needles in elevated CO2 concentration in comparison with sun needles in ambient CO2 concentration. In conclusion, it was demonstrated that the first layer of mesophyll is not always representative for the needle cross section. If technically feasible, SUR is recommended for analysis of chloroplast ultrastructure. The simplified sampling design can be applied, e.g., for comparisons of many different treatments. However, it should be combined with other approaches to characterize the chloroplast function and the results carefully considered and interpreted.

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.

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.

This chapter gives examples of basic procedures of quantification of plant structures with the use of image analysis, which are commonly employed to describe differences among experimental treatments or phenotypes of plant material. Tasks are demonstrated with the use of ImageJ, a widely used public domain Java image processing program. Principles of sampling design based on systematic uniform random sampling for quantitative studies of anatomical parameters are given to obtain their unbiased estimations and simplified "rules of thumb" are presented. The basic procedures mentioned in the text are (1) sampling, (2) calibration, (3) manual length measurement, (4) leaf surface area measurement, (5) estimation of particle density demonstrated on an example of stomatal density, and (6) analysis of epidermal cell shape.

Our goal was to find an optimal tissue clearing protocol for whole-mount imaging of embryonic and adult hearts and whole embryos of transgenic mice that would preserve green fluorescent protein GFP fluorescence and permit comparison of different currently available 3D imaging modalities. We tested various published organic solvent- or water-based clearing protocols intended to preserve GFP fluorescence in central nervous system: tetrahydrofuran dehydration and dibenzylether protocol (DBE), SCALE, CLARITY, and CUBIC and evaluated their ability to render hearts and whole embryos transparent. DBE clearing protocol did not preserve GFP fluorescence; in addition, DBE caused considerable tissue-shrinking artifacts compared to the gold standard BABB protocol. The CLARITY method considerably improved tissue transparency at later stages, but also decreased GFP fluorescence intensity. The SCALE clearing resulted in sufficient tissue transparency up to ED12.5; at later stages the useful depth of imaging was limited by tissue light scattering. The best method for the cardiac specimens proved to be the CUBIC protocol, which preserved GFP fluorescence well, and cleared the specimens sufficiently even at the adult stages. In addition, CUBIC decolorized the blood and myocardium by removing tissue iron. Good 3D renderings of whole fetal hearts and embryos were obtained with optical projection tomography and selective plane illumination microscopy, although at resolutions lower than with a confocal microscope. Comparison of five tissue clearing protocols and three imaging methods for study of GFP mouse embryos and hearts shows that the optimal method depends on stage and level of detail required.

Vasa vasorum supply both the tunica adventitia and the tunica media of major arteries with nutrients and oxygen. We estimated the density of von Willebrand factor-positive profiles of vasa vasorum visible in transversal histological sections of 123 tissue samples collected from five anatomical positions in the porcine aortae of growing pigs (n=25). The animals ranged in age from 0 to 230 days. The tunica media of the thoracic aorta had a greater vasa vasorum density, with microvessels penetrating deeper towards the lumen than in the abdominal aorta. The density of vasa vasorum gradually decreased with age in both the media and the adventitia. The relative depth into which the vasa vasorum penetrated and where they branched remained constant during the ageing and growth of the media. The ratio of the tunica media and tunica adventitia thicknesses did not change in the single aortic segments during ageing. The media of older animals received fewer but equally distributed vasa vasorum. A greater density of vasa vasorum in the media was correlated with greater media thickness and a greater elastin fraction (data on elastin taken from another study on the same samples). Immunohistochemical quantification revealed deeper penetration of vasa vasorum towards the adluminal layers of the tunica media that were hitherto reported to be avascular. The complete primary morphometric data, in the form of continuous variables, have been made available as a supplement. Mapping of the vasa vasorum profile density and position has promising illustrative potential for studies on atherosclerotic and inflammatory neovascularization, aortic aneurysms, and drug distribution from arterial stents in experimental porcine models.

Image analysis methods are widely used tools for an objective pilling evaluation. However, due to their intrinsic limitations, simple image analysis of digital photographs is not a suitable procedure for the pilling evaluation of fabrics with complex structures and distinct patterns. Generally, a fabric pattern or a complex fabric structure have presented significant problem to the pills detection. This paper describes a system of the objective pilling evaluation of unicolor fabrics and even fabrics with complex patterns. The main points of the pilling evaluation system include a 3D fabric surface reconstruction from shading based on gradient field method, using image analysis tools for the pills detection and an estimation of an objective pilling grade. In order to enable the estimation of the objective pilling grade, a relationship between the pilling grades obtained by a subjective method and selected pill characteristics was specified using a linear regression model. The method was tested on a set of ...

Fluorescence recovery after photobleaching (FRAP) is the preferred method for analyzing the lateral mobility of fluorescently-tagged proteins in the plasma membranes (PMs) of live cells. FRAP experiments are described as being easy to perform; however, the analysis of the acquired data can be difficult. The evaluation procedure must be properly combined with the imaging setup of the confocal microscope to provide unbiased results. With the aim of increasing the accuracy of determining the diffusion coefficient (D) and mobile fraction (Mf) of PM proteins, we developed a novel method for FRAP analysis in the equatorial plane of the cell. This method is based on the calculation of photobleaching characteristics, derived from the light intensity profile and optical parameters of the confocal microscope, and on the model of fluorescent molecule diffusion in PM regions outside of the focal plane. Furthermore, cell movement artifacts in the FRAP data are ameliorated by using a region of interest, which is not fixed but instead moves adaptively in coordination with the movement of cells. When this method was used to determine the mobility of the δ-opioid receptor-eYFP in HEK293 cells, a highly significant decrease in receptor mobility was detected in cholesterol-depleted cells. This decrease was fully reversible by the replenishment of cholesterol levels. Our results demonstrate the crucial role played by cholesterol in the dynamic organization of δ-opioid receptors in the PM under in vivo conditions. Our method may be applied for the determination of the D and Mf values of other PM proteins.

Most embryonic ventricular cardiomyocytes are quite uniform, in contrast to the adult heart, where the specialized ventricular conduction system is molecularly and functionally distinct from the working myocardium. We thus hypothesized that the preferential conduction pathway within the embryonic ventricle could be dictated by trabecular geometry. Mouse embryonic hearts of the Nkx2.5:eGFP strain between ED9.5 and ED14.5 were imaged whole mount after clearing by confocal microscopy, and reconstructed in 3D at 3.4 µm isotropic voxel size. The local orientation of the trabeculae, responsible for the anisotropic spreading of the signal, was characterized using spatially homogenized tensors (3x3 matrices) calculated from the trabecular skeleton. Activation maps were simulated assuming constant speed of spreading along the trabeculae. The results were compared with experimentally obtained epicardial activation maps generated by optical mapping with a voltage-sensitive dye. Simulated impulse propagation starting from the top of interventricular septum revealed the first epicardial breakthrough at the interventricular grove, similar to experimentally obtained activation maps. Likewise, ectopic activation from the left ventricular base perpendicular to dominant trabecular orientation resulted in isotropic and slower impulse spreading on the ventricular surface in both simulated and experimental conditions. We conclude that in the embryonic pre-septation heart, the geometry of the A-V connections and trabecular network is sufficient to explain impulse propagation and ventricular activation patterns.

Here we investigated the effect of disruption of plasma membrane integrity by cholesterol depletion on thyrotropin-releasing hormone receptor (TRH-R) surface mobility in HEK293 cells stably expressing TRH-R-eGFP fusion protein (VTGP cells). Detailed analysis by fluorescence recovery after photobleaching (FRAP) in bleached spots of different sizes indicated that cholesterol depletion did not result in statistically significant alteration of mobile fraction of receptor molecules (Mf). The apparent diffusion coefficient (Dapp) was decreased, but this decrease was detectable only under the special conditions of screening and calculation of FRAP data. Analysis of mobility of receptor molecules by raster image correlation spectroscopy (RICS) did not indicate any significant difference between control and cholesterol-depleted cells. Results of our FRAP and RICS experiments may be collectively interpreted in terms of a “membrane fence” model which regards the plasma membrane of living cells as compartmentalized plane where lateral diffusion of membrane proteins is limited to restricted areas by cytoskeleton constraints. Hydrophobic interior of plasma membrane, studied by steady-state and time-resolved fluorescence anisotropy of hydrophobic membrane probe DPH, became substantially more “fluid” and chaotically organized in cholesterol-depleted cells. Decrease of cholesterol level impaired the functional coupling between the receptor and the cognate G proteins of Gq/G11 family. In conclusion: the presence of an unaltered level of cholesterol in the plasma membrane represents an obligatory condition for an optimum functioning of TRH-R signaling cascade. The decreased order and increased fluidity of hydrophobic membrane interior suggest an important role of this membrane area in TRH-R–Gq/G11α protein coupling.

The effect of ageing on the capillary network in skeletal muscles has produced conflicting results in both, human and animals studies. Some of the inconsistencies are due to non-comparable and biased methods that were applied on thin transversal sections, especially in muscles with complicated morphological structures, such as in human masseter muscle. We present a new immunohistochemical method for staining capillaries and muscle fibres in 100 µm thick sections as well as novel approach to 3D visualization of capillaries and muscle fibres. Applying confocal microscopy and virtual 3D stereological grids, or tracing capillaries in virtual reality, length of capillaries within a muscle volume or length of capillaries adjacent to muscle fibre per fibre length, fibre surface or fibre volume were evaluated in masseter muscle of young and old subjects by an unbiased approach. Our findings show that anatomic capillarity is well maintained in masseter muscle in old subjects; however, vascular remodelling occurs with age, which could be a response to changed muscle function and age-related muscle fibre type transformations.

This paper presents 3D reconstruction of fabric surface with the pills, detection of the pills and qualitative evaluation of pilling with the help of spatial data analysis. 3D reconstruction of fabric surface is performed with the help of gradient fields method. The surface pills are extracted from 3D image by techniques of image analysis. Consequently, the tools of spatial data analysis as K function, density and test are used for experiment of qualitative evaluation of pills distribution in the space.

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.

Terrestrial isopods moult first the posterior and then the anterior half of the body, allowing for storage and recycling of CaCO3. We used synchrotron-radiation microtomography to estimate mineral content within skeletal segments in sequential moulting stages of Porcellio scaber. The results suggest that all examined cuticular segments contribute to storage and recycling, however, to varying extents. The mineral within the hepatopancreas after moult suggests an uptake of mineral from the ingested exuviae. The total maximum loss of mineral was 46% for the anterior and 43% for the posterior cuticle. The time course of resorption of mineral and mineralisation of the new cuticle suggests storage and recycling of mineral in the posterior and anterior cuticle. The mineral in the anterior pereiopods decreases by 25% only. P. scaber has long legs and can run fast; therefore, a less mineralised and thus lightweight cuticle in pereiopods likely serves to lower energy consumption during escape behaviour. Differential demineralisation occurs in the head cuticle, in which the cornea of the complex eyes remains completely mineralised. The partes incisivae of the mandibles are mineralised before the old cuticle is demineralised and shed. Probably, this enables the animal to ingest the old exuviae after each half moult.

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.

Confocal microscopy is a suitable method for measurements and visualization of skeletal muscle fibres and the neighbouring capillaries. When using 3D images of thick sections the tissue deformation effects should be avoided. We studied the deformation in thick sections of the rat skeletal muscle from complete stacks of images captured with confocal microscope. We measured the apparent thickness of the stacks and compared it to the slice thickness deduced from calibrated microtome settings. The ratio of both values yielded the axial scaling factor for every image stack. Careful sample preparation and treatment of the tissue cryosections with cold Ringer solution minimize the tissue deformation. We conclude that rescaling by the inverse of the axial scaling factor of the stack of optical slices in the direction of the microscope optical axis satisfactorily corrects the axial deformation of skeletal muscle samples.

Three-dimensional analyses of the spatial arrangement, spatial orientation and preferential directions of systems of fibers are frequent tasks in many scientific fields, including the textile industry, plant biology and tissue modeling. In biology, systems of oriented and branching lines are often used to represent the three-dimensional directionality and topology of microscopic blood vessels supplying various organs. In our study, we present a novel p(χ2) (chi-square) method for evaluating the anisotropy of line systems that involves comparing the observed length densities of lines with the discrete uniform distribution of an isotropic line system with the χ2-test. Using this method in our open source software, we determined the rose of directions, preferential directions and level of anisotropy of linear systems representing the microscopic blood vessels in samples of various regions from human brains (cortex, subcortical gray matter and white matter). The novel method was compared with two other methods used for anisotropy quantification (ellipsoidal and fractional anisotropy). All three methods detected different levels of anisotropy of blood microvessels in human brain. The microvascular bed in the cortex was closer to an isotropic network, while the microvessels supplying the white matter appeared to be an anisotropic and direction-sensitive system. All three methods were able to determine the differences between various brain regions. The advantage of our p(χ2) method is its high correlation with the number of preferential directions of the line system. However, the software, named esofspy, is able to calculate all three of the measures of anisotropy compared and documented in this paper, thus making the methods freely available to the scientific community.

Image analysis techniques for preprocessing, segmentation and estimation of geometrical characteristics of fiber-like structures from 2-D or 3-D images captured by a confocal microscope are presented. Methods are demonstrated on fiber-like biological structure: endoplasmic reticulum (ER) of tobacco cells. In the presented analysis of 2-D images of ER before and after the treatment of latrunculin B, ER and ER tubules were segmented and the area density of ER as well as the length density of ER tubules in the cell cortical layer were estimated by automatic image analysis algorithms. Images of 3-D arrangement of ER were reconstructed and rendered by various visualization techniques.

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.

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.

Activation of mast cells by aggregation of the high-affinity IgE receptors (FcεRI) initiates signaling events leading to the release of inflammatory and allergic mediators stored in cytoplasmic granules. A key role in this process play changes in concentrations of intracellular Ca2+ controlled by store-operated Ca2+ entry (SOCE). Although microtubules are also involved in the process leading to degranulation, the molecular mechanisms that control microtubule rearrangement during activation are largely unknown. In this study, we report that activation of bone marrow-derived mast cells (BMMCs) induced by FcεRI aggregation or treatment with pervanadate or thapsigargin results in generation of protrusions containing microtubules (microtubule protrusions). Formation of these protrusions depended on the influx of extracellular Ca2+. Changes in cytosolic Ca2+concentration also affected microtubule plus-end dynamics detected by microtubule plus-end tracking protein EB1. Experiments with knockdown or reexpression of STIM1, the key regulator of SOCE, confirmed the important role of STIM1 in the formation of microtubule protrusions. Although STIM1 in activated cells formed puncta associated with microtubules in protrusions, relocation of STIM1 to a close proximity of cell membrane was independent of growing microtubules. In accordance with the inhibition of Ag-induced Ca2+ response and decreased formation of microtubule protrusions in BMMCs with reduced STIM1, the cells also exhibited impaired chemotactic response to Ag. We propose that rearrangement of microtubules in activated mast cells depends on STIM1-induced SOCE, and that Ca2+ plays an important role in the formation of microtubule protrusions in BMMCs.

We used immunochemistry, light microscopy and stereological methods for quantitative description of the microvascular network in 13 tissue samples of the human brain. While the tortuosity of microvessels was comparable in all brain parts under study, the length density of microvessels was higher in subcortical grey matter (652.5±162.0 mm-2) and in the cortex (570.9±71.8 mm-2) than in the white matter (152.7±42.0 mm-2). The numerical density of microvessels was higher in subcortical grey matter (3782.0±1602.0 mm-3) and cerebral cortex (3160.0±638.4 mm-3) than in white matter (627.7±318.5 mm-3). We developed simulation software gensei which generates series of images representing three-dimensional models of microvessels with known length density, volume fraction, and surface density. The simulations are statistically similar to real microvessel networks and can be used for computer modelling of brain perfusion.

Nuclear actin and nuclear myosin I (NMI) are important players in transcription of ribosomal genes. Transcription of rDNA takes place in highly organized intranuclear compartment, the nucleolus. In this study, we characterized the localization of these two proteins within the nucleolus of HeLa cells with high structural resolution by means of electron microscopy and gold-immunolabeling. We demonstrate that both actin and NMI are localized in specific compartments within the nucleolus, and the distribution of NMI is transcription-dependent. Moreover, a pool of NMI is present in the foci containing nascent rRNA transcripts. Actin, in turn, is present both in transcriptionally active and inactive regions of the nucleolus and colocalizes with RNA polymerase I and UBF. Our data support the involvement of actin and NMI in rDNA transcription and point out to other functions of these proteins in the nucleolus, such as rRNA maturation and maintenance of nucleolar architecture.

The purpose of this study was to compare two electron microscopy embedding media – LR White and Unicryl – with regard to cell morphologyical and immunohistochemical preservation properties for the study of fixation-sensitive nuclear antigens. Human cervical carcinoma (HeLa) cells were fixed with 2% paraformaldehyde and 0.1% glutaraldehyde, and embedded in parallel in the two resins: LR White and Unicryl using; two different polymerization protocols were used for each resin. Preservation of fine nuclear structure was good after LR White and poor after Unicryl embedding. Immunogold labeling of Sm antigen was significantly stronger on LR White sections. Polymerization by UV light resulted in stronger and more specific labeling than heat polymerization. These results show that LR White is advantageous over Unicryl for the study of nuclear antigens requiring delicate aldehyde fixation.

Three consecutive pairs of head cavities (premandibular, mandibular, and hyoid) found in elasmobranchs have been considered as remnants of preotic 'head' somites-serial homologues of the myotomic compartments of trunk somites that give rise to the extraoccular musculature. Here, we study a more derived vertebrate, and show that cavitation is more complex in the head of Crocodylus niloticus, than just the occurrence of three pairs of cavities. Apart from the premandibular cavities, paired satellite microcavities, and unpaired extrapremandibular microcavities are recognized in the prechordal region as well. We observed that several developmental phenomena occur at the same time as the formation of the head cavities (premandibular, satellite, extrapremandibular, mandibular, and hyoid) appear temporarily in the crocodile embryo. These are 1) rapid growth of the optic stalk and inflation of the optic vesicle; 2) release of the intimate topographical relationships between the neural tube, notochord and oral gut; 3) tendency of the prechordal mesenchyme to follow the curvature of the forebrain; and 4) proliferation of the prechordal mesenchyme. On the basis of volumetric characters, only the hyoid cavity and hyoid condensation is comparable to the trunk somitocoel and somite, respectively.

Summary Although kidney trauma is a relatively common injury, its microscopic biomechanics are poorly understood. Experimental low-grade trauma in pig kidneys was studied using optical microscopy. We observed ruptures in the cortex as well as in the medulla. Both parts of the renal parenchyma were damaged, even in areas of the kidneys that were free of macroscopic cracks on the surface. To determine which constituents of the renal cortex and medulla, i.e. tubular parts of the nephron or the interstitial connective tissue, were less resistant to injury during the drop shatter test, we applied a simple stereological method to discriminate between random and tissue-specific rupture propagation. The ruptures propagated predominantly through the interstitial connective tissue of the renal cortex and medulla. The volume fraction of the tubules assessed by the Cavalieri principle was 90.4% within the renal cortex and 52.4% within the medulla. The most frequently affected blood vessels were the arcuate and interlobular veins, followed by the arcuate and interlobular arteries. No disruptions of the renal calyces were found.

We used mechanical measurements and unbiased statistical morphometry in order to supply the computer model of smooth muscle cells (SMC) and collagen connective tissue of a gastropod Arion sp. with sufficient input data. We identified the elasticity modulus for both living (34 kPa) and formalin-fixed samples (440 kPa). The relative volume proportion of SMC was 57.8%, their numerical density was 425 925 mm-3, surface density of SMC was 405.9 mm-1 and the mean cell volume was 1 358 .m3.

We postulated that, in rat extensor digitorum longus muscle (EDL), the length of capillaries per fibre surface area (Lcap/Sfib) and per fibre volume (Lcap/Vfib) could reflect fibre-type transformations accompanied by changes in oxidative metabolic profile and selective fibre-type atrophy. We excised rat EDL muscle 2 weeks after the sciatic nerve was cut (acute denervation; DEDL) and 4 weeks after the nerve was crushed (early reinnervation; REDL) and characterised muscle fibre-type transformation by the expression of myosin heavy-chain isoforms and by succinate dehydrogenase (SDH) and nicotinoamide adenine dinucleotide-tetrazolium reductase (NADH-TR) reactions. The numerical percentage (N/N) and area percentage (A/A) of pure and hybrid fibres and their diameter were determined, as was the A/A of SDH- and NADH-TR-positive fibres. The length of capillaries per fibre length (Lcap/Lfib), Lcap/Sfib and Lcap/Vfib were estimated in REDL and Lcap/Vfib in DEDL. In DEDL, the type 2x and 2b fibres evidently atrophied, with the N/N of type 2x fibres being lower and that of hybrid fibres higher. In REDL, the N/N of hybrid fibres was even higher, consequent to a lower N/N of type 2b fibres; however, fibre diameters approached values of the control EDL. Compared with control EDL, denervated and reinnervated muscles exhibited a higher A/A of oxidative fibres. This is probably the result of fibre-type transformation and selective fibre atrophy. We conclude that capillary length does not change during acute denervation and early reinnervation. The obtained higher values of Lcap/Sfib and Lcap/Vfib are related to changes in muscle fibre cross-sectional area.

Recent design-based stereological methods that can be applied to thick sections cut in an arbitrary direction are presented and their implementation for measuring mesophyll anatomical characteristics is introduced. These methods use software-randomized virtual 3D probes, such as disector and fakir test probes, in stacks of optical sections acquired using confocal microscopy. They enable unbiased estimations of the mean mesophyll cell volume, mesophyll cell number in a needle, and for the first time an internal surface area of needles or other narrow leaves directly from the fresh tissue cross-sections cut using a hand microtome. Therefore, reliable results can be obtained much faster than when using a standard microtechnical preparation. The proposed methods were tested on Norway spruce needles affected for 1 year by acid rain treatment. The effect of acid rain resulted in changes of mesophyll parameters: the ratio of intercellular spaces per mesophyll cell volume increased, while needle internal surface area, total number of mesophyll cells, and number of mesophyll cells per unit volume of a needle decreased in the treated needles.

Background In the past few years, computer-based analysis of atomic-force microscopic images has acquired increasing importance for studying biomolecules such as DNA. On the one hand, fully automated methods do not allow analysis of complex shapes; on the other hand, manual methods are usually time consuming and inaccurate. The semiautomated approach presented in this report overcomes the drawbacks of both methods. Methods Two kinds of images were analyzed: computer-generated filaments that modeled circular DNA molecules on a surface and real atomic-force microscopic images of DNA molecules adsorbed on an appropriate substrate surface. Results The algorithm was tested on a group of 140 simulated and 189 real plasmids with a nominal length of 913 nm. The accuracy of the length measurement was statistically evaluated on the ensemble of molecules, with particular attention to the influence of the noise. Mean contour lengths of 912 ± 5 nm and 910 ± 47 nm were found for simulated and real plasmids, respectively. The measured end-to-end distance of λ-DNA molecules as a function of their contour length is reported, from which it is possible to estimate the stiffness of the DNA molecules adsorbed onto a surface; the value obtained for the DNA persistence length (42 ± 5 nm) is consistent with values measured by other imaging techniques. Conclusions An interactive algorithm for DNA molecule measurements based on the detection of the filament ridge line in a digitized image is presented. The simulation of artificial filaments combined with the experimental data demonstrates that the proposed method can be a valuable tool for the DNA contour length evaluation, especially in the case of complex shapes where the use of automatic methods is not possible. © 2005 Wiley-Liss, Inc.

Ultrastructural identification of subcellular morphologically inconspicuous compartments is based on detection of specific molecules or by a presence of specific functions. Such compartments are detected using antibodies with attached label, usually gold particles. However, the gold particles have a point pattern, while a compartment is a coherent area. In addition, some background labeling is always present that complicates identification of the labeled compartments. The aim of this study was therefore to develop a stereological method that would enable us to define cellular compartments based on delineating the borders of gold particle clusters, and to test the practical use of the method using biological experimental data. New computer program plug-ins were developed to facilitate the practical use of the stereological method. The kernel estimation method was successfully tested by detection of ribosomal rRNA over morphologically recognizable nucleoli. In a next step, we successfully detected individual chromosomal domains-nuclear compartments that cannot be distinguished in cell nuclei morphologically. The results show that the new stereological/image analysis method is well able to discriminate cellular compartments based on density of immunogold particles. The plug-ins were made available to scientific community at http://nucleus.biomed.cas.cz/gold.

The confocal microscope can image a specimen in its natural environment forming a 3D image of the whole structure by scanning it and collecting light through a small aperture (pinhole), allowing in vivo and in vitro observations. So far, the confocal fluorescence microscope (CFM) is considered a true volume imager because of the role of the pinhole that rejects information coming from out-of-focus planes. Unfortunately, intrinsic imaging properties of the optical scheme presently employed yield a corrupted image that can hamper quantitative analysis of successive image planes. By a post-image collection restoration, it is possible to obtain an estimate, with respect to a given optimization criterium, of the true object, utilizing the impulse response of system or Point Spread Function (PSF). The PSF can be measured or predicted so as to have a mathematical and physical model of the image-formation process. Further modelling and recording noise as an additive Gaussian process has used the regularized Iterative Constrained Tykhonov Miller (ICTM) restoration algorithm for solving the inverse problem. This algorithm finds the best estimate iteratively searching among the possible positive solutions; in the Fourier domain, such an approach is relatively fast and elegant. In order to compare the effective improvement in the quantitative image information analysis, we measured the volume of reference objects before and after image restoration, using the isotropic Fakir method.