Development of ventricular trabeculae affects electrical conduction in the early endothermic heart.

BACKGROUND: The ventricular trabeculae play a role, among others, in the impulse spreading in ectothermic hearts. Despite the morphological similarity with the early developing hearts of endotherms, this trabecular function in mammalian and avian embryos was poorly addressed. RESULTS: We simulated impulse propagation inside the looping ventricle and revealed delayed apical activation in the heart with inhibited trabecular growth. This finding was corroborated by direct imaging of the endocardial surface showing early activation within the trabeculae implying preferential spreading of depolarization along with them. Targeting two crucial pathways of trabecular formation (Neuregulin/ErbB and Nkx2.5), we showed that trabecular development is also essential for proper conduction patterning. Persistence of the slow isotropic conduction likely contributed to the pumping failure in the trabeculae-deficient hearts. CONCLUSIONS: Our results showed the essential role of trabeculae in intraventricular impulse spreading and conduction patterning in the early endothermic heart. Lack of trabeculae leads to the failure of conduction parameters differentiation resulting in primitive ventricular activation with consequent impact on the cardiac pumping function.

Horizontal deformation of skeletal muscle thick sections visualised by confocal microscopy.

Certain morphological parameters of the skeletal muscle tissue can be better understood via 3D considerations. Fluorescent confocal microscopy of thick tissue sections is a well-established method for visualising and measuring skeletal muscle fibres and surrounding capillaries in 3D. However, thick tissue sections are prone to deformations which may significantly influence some stereological and morphometric results like muscle fibre diameter and capillary length, but not dimensionless parameters like object number and Euler-Poincaré characteristics. To better understand this phenomenon, we studied the horizontal deformation of thick (100 µm) transverse skeletal muscle sections, by comparing the muscle fibre diameters measured on thick sections to muscle fibre diameters measured on thin (10 µm) sections of the same sample. Diameter changes were further correlated with shrinkage in the Z direction (axial shrinkage) and deviation of the muscle fibre preferential axis from the Z-axis. We showed that the thick sections dilated in horizontal and shrunk in Z direction, and that the magnitude of horizontal dilation was associated with the magnitude of shrinkage in the Z direction. The latter was more pronounced in transversely than obliquely cut tissue sections. The results emphasise that even when shrinkage in the Z direction can be corrected using calibration, it is important to optimise histological protocols to minimise the Z-axis collapse that could cause horizontal dilation. LAY DESCRIPTION: In skeletal muscle research, 3D analysis is especially important for studying the microvasculature. Laser scanning confocal microscopy of skeletal muscle thick tissue sections is a well-established method for visualising and measuring skeletal muscle fibres and surrounding capillaries in 3D. However, such sections are prone to deformations which may significantly influence the study results. To better understand this phenomenon, we studied the horizontal deformation of thick transverse skeletal muscle sections. We compared the average muscle fibre diameters measured on thick skeletal muscle sections, thin fixed skeletal muscle sections and immunohistochemically stained thin skeletal muscle sections with the muscle fibre diameters measured on thin native skeletal muscle sections of the same sample, with the latter condition serving as the standard diameters (ie the control condition). We further studied the association among muscle fibre diameter changes, shrinkage of the thick skeletal muscle sections in the Z direction and their sectioning angle. We showed that the thick skeletal muscle sections dilated in the horizontal direction and shrunk in the Z direction, and that the magnitude of horizontal dilation was associated with the magnitude of shrinkage in Z direction. The shrinkage in the Z direction was more pronounced in transversely than obliquely cut tissue sections. These results emphasise that even when shrinkage in the Z direction can be corrected using Z-axis calibration, it is very important to optimise histological protocols to minimise the Z-axis collapse that could cause horizontal dilation in order to enhance the integrity of study results.

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.

Calcaneal Tendon Collagen Fiber Morphometry and Aging.

Fibrillar collagen in tendons and its natural development in rabbits are discussed in this paper. Achilles tendons from newborn (~7 days) to elderly (~38 months) rabbits were monitored in intact (n tendons=24) and microtome sectioned (n tendons=11) states with label-free second harmonic generation microscopy. After sectioning, the collagen fiber pattern was irregular for the younger animals and remained oriented parallel to the load axis of the tendon for the older animals. In contrast, the collagen fiber pattern in the intact samples followed the load axis for all the age groups. However, there was a significant difference in the tendon crimp pattern appearance between the age groups. The crimp amplitude (A) and wavelength (Λ) started at very low values (A=2.0±0.6 µm, Λ=19±4 µm) for the newborn animals. Both parameters increased for the sexually mature animals (>5 months old). When the animals were fully mature the amplitude decreased but the wavelength kept increasing. The results revealed that the microtome sectioning artifacts depend on the age of animals and that the collagen crimp pattern reflects the physical growth and development.

TRH-receptor mobility and function in intact and cholesterol-depleted plasma membrane of HEK293 cells stably expressing TRH-R-eGFP.

UNLABELLED: 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.

Comparison of different tissue clearing methods and 3D imaging techniques for visualization of GFP-expressing mouse embryos and embryonic hearts.

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.

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.

STIM1-directed reorganization of microtubules in activated mast cells.

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 Ca(2+) controlled by store-operated Ca(2+) 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 Ca(2+). Changes in cytosolic Ca(2+)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 Ca(2+) 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 Ca(2+) plays an important role in the formation of microtubule protrusions in BMMCs.

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.

Skeletal muscle myosin heavy chain expression and 3D capillary network changes in streptozotocin-induced diabetic female mice.

It is not well-understood how type 1 diabetes (T1DM) affects skeletal muscle histological phenotype, particularly capillarisation. This study aimed to analyze skeletal muscle myosin heavy chain (MyHC) fibre type changes and 3D capillary network characteristics in experimental T1DM mice. Female C57BL/6J-OlaHsd mice were categorized into streptozotocin (STZ)-induced diabetic (n = 12) and age-matched non-diabetic controls (n =12). The muscle fibre phenotype of the soleus, gluteus maximus, and gastrocnemius muscles were characterized based on the expression of MyHC isoforms, while capillaries of the gluteus maximus were assessed with immunofluorescence staining, confocal laser microscopy and 3D image analysis. STZ-induced diabetic mice exhibited elevated glucose levels, reduced body weight, and prolonged thermal latency, verifying the T1DM phenotype. In both T1DM and non-diabetic mice, the gluteus maximus and gastrocnemius muscles predominantly expressed fast-twitch type 2b fibers, with no significant differences noted. However, the soleus muscle in non-diabetic mice had a greater proportion of type 2a fibers and comparable type 1 fiber densities (26.2 ± 14.6% vs 21.9 ± 13.5%) relative to diabetic mice. T1DM mice showed reduced fiber diameters (P = 0.026), and the 3D capillary network analysis indicated a higher capillary length per muscle volume in the gluteus maximus of diabetic mice compared to controls (P < 0.05). Overall, T1DM induced significant changes in the skeletal muscle, including shifts in MyHC fibre types, decreased fibre diameters, and increased relative capillarisation, possibly due to muscle fibre atrophy. Our findings emphasize the superior detail provided by the 3D analytical method for characterizing skeletal muscle capillary architecture, highlighting caution in interpreting 2D data for capillary changes in T1DM.

Anguimorpha as a model group for studying the comparative heart morphology among Lepidosauria: Evolutionary window on the ventricular septation.

The group Anguimorpha represents one of the most unified squamate clades in terms of body plan, ecomorphology, ecophysiology and evolution. On the other hand, the anguimorphs vary between different habitats and ecological niches. Therefore, we focused on the group Anguimorpha to test a possible correlation between heart morphology and ecological niche with respect to phylogenetic position in Squamata with Sphenodon, Salvator, and Pogona as the outgroups. The chosen lepidosaurian species were investigated by microCT. Generally, all lepidosaurs had two well-developed atria with complete interatrial septum and one ventricle divided by ventricular septa to three different areas. The ventricles of all lepidosaurians had a compact layer and abundant trabeculae. The compact layer and trabeculae were developed in accordance with particular ecological niche of the species, the trabeculae in nocturnal animals with low metabolism, such as Sphenodon, Heloderma or Lanthanotus were more massive. On the other hand athletic animals, such as varanids or Salvator, had ventricle compartmentalization divided by three incomplete septa. A difference between varanids and Salvator was found in compact layer thickness: thicker in monitor lizards and possibly linked to their mammalian-like high blood pressure, and the level of ventricular septation. In summary: heart morphology varied among clades in connection with the ecological niche of particular species and it reflects the phylogenetic position in model clade Anguimorpha. In the absence of fossil evidence, this is the closest approach how to understand heart evolution and septation in clade with different cardiac compartmentalization levels.

Chromosomal dynamics of cell cycle regulator gene p21 during transcriptional activation.

The radial position of a gene within its chromosome territory (CT) in the interphase nucleus is thought to depend on the transcriptional activity of the gene and on transcriptional activity, gene density, and conformation of the chromosomal surrounding. In this study we analyzed the position of the cell cycle regulator gene p21 within the CT of human chromosome 6 (HSA6) upon transcriptional activation. Whereas the majority of active p21 genes is located in the interior of the CT of HSA6, induction of p21 transcription correlates with increased variation of gene localization within the CT and with a higher percentage of p21 genes located at the periphery of the CT. Additionally it demonstrates once more that transcription can take place throughout CTs. Comparison of the p21 locus with two non-coding regions on HSA6 showed that both non-coding sequences are located more frequently in the interior of the CT than p21 genes although they are situated in chromosomal neighborhoods with widely differing gene density and regional transcriptional activity. Thus our data support models describing an influence of the transcriptional activity of a gene on the localization within its CT. However, our data also indicate that additional factors such as chromatin remodeling are implicated in the positioning of genes within the respective chromosome territory.

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.

How to asses, visualize and compare the anisotropy of linear structures reconstructed from optical sections--a study based on histopathological quantification of human brain microvessels.

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.

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.

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.

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.

Determination of δ-opioid receptor molecules mobility in living cells plasma membrane by novel method of FRAP analysis.

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.