Serial Optical Sections Research Articles

91 A NOVEL TECHNIQUE FOR EVALUATION OF THE LIPID CONTENT IN PIG EMBRYOS

A high level of lipids, mainly triglycerides and fatty acids, present in embryo cells in the form of lipid droplets is the major factor associated with low cryopreservation of porcine embryos. Previous results demonstrated that the low tolerance of pig embryos to cryopreservation can be increased through reduction of lipid droplet contents. Therefore, in order to improve cryopreservation techniques of porcine embryos, it is fundamental to establish proper culture conditions which ultimately will enable a decrease in lipid content. Unfortunately, there are no precise and efficient methods to evaluate the lipid contents of single pig embryos. Previously used stereological analysis combined with physical serial sectioning (Romek et al. 2007 Reprod. Domest. Anim. in press) is time-consuming, and measurement of triglyceride levels based on enzymatic hydrolysis eliminates other types of lipids from the analysis. Taking the above problems into account, we have developed a new method for evaluation of total lipid content in pig embryos. It is based on visualization of lipid droplets using the specific fluorescent dye Nile red and applying confocal scanning microscopy followed by the Cavalieri method. This method enables measurement of several stereological parameters, especially the volume density of lipid droplets per unit volume of cytoplasm Vv(fat,c), which quantifies most precisely the amount of intracellular lipid. The experiment was carried out on 2- to 4-cell and 8- to 16-cell pig embryos, morulae, blastocysts, and late blastocysts cultured in vitro. Embryos were developed from in vivo-produced zygotes to appropriate stages of development in North Carolina State University (NCSU) 23 medium. For each stage, ten of the embryos were examined. Embryos were denuded and fixed with 2% glutaraldehyde and 2% formaldehyde, stained with 100 nm Nile red (Molecular Probes, Leiden, The Netherlands), and analyzed by means of a confocal microscope LSM 510 Meta (Carl Zeiss MicroImaging GmbH, G�ttingen, Germany). Serial optical sections of each individual embryo were measured by the point counting method, and then the Cavalieri method was used to estimate Vv(fat,). Vv(fat,c) values calculated for embryos at different stages of development were compared by one-way analysis of variance and Tukey's intervals. For cultured pig embryos, volume density of lipid droplets Vv(fat,c) significantly decreased during cleavage from 0.55 µm3 µm–3 at the 2- to 4-cell-embryo stage to 0.46 µm3 µm–3 at the blastocyst stage. The differences between lipid droplet volumes calculated for morulae, blastocysts, and late blastocysts were statistically significant. In conclusion, our new method is more precise, efficient, and quick in comparison to previously used ones. Moreover, we confirmed that the content of total lipids in cultured pig embryo is reduced during its development. This research was funded by the State Committee for Scientific Research (Project No. 2 P06D 003 26) and Net of Reproduction Biotechnology.

Attenuation of microtubule associated protein-2 degradation after mild head injury by mexiletine and calpain-2 inhibitor

The objective of the study was to address the early effects of mild, closed, head injuries on neuronal stability and the prevention of microtubule-associated protein-2 (MAP-2) degradation by mexiletine and calpain-2 inhibitor. Twenty-four rats were divided into four groups: control group (1); trauma group without treatment (2); mexiletine-pretreated and subjected to trauma group (3); trauma subjected and then calpain-2 inhibitor received group (4). All animals were subjected to mild, closed, head trauma. Frontal lobes were removed and processed for staining and immunofluorescent labelling of MAP-2 cytoskeletal proteins, which were evaluated by confocal microscopy in serial optical sections showing the three dimensional cytoarchitecture of affected areas. MAP-2 decoration in almost all neurons obtained from traumatized brain regions drastically diminished, while minute filamentous and granular profiles in axons and/or dendrites were retained together implying a massive degradation/depolymerization of microtubules. In contrast, in mexiletine-pretreated animals, MAP-2 positivity in axonal and perikaryonal profiles was fairly retained, which clearly depicts the protective role of mexiletine after trauma. Compared with mexiletine-pretreated group, calpain-2 inhibitor treated group displayed a less well-preserved MAP-2 expression. Mexiletine can prevent cytoskeletal structure and protein degradation after mild head trauma. Calpain-2 inhibitor prevents protein degradation, but cytoskeletal organization is better preserved with mexiletine.

Essential Role of the Apoptotic Cell Engulfment Genes draper and ced-6 in Programmed Axon Pruning during Drosophila Metamorphosis

Axon pruning is a common phenomenon in neural circuit development. Previous studies demonstrate that the engulfing action of glial cells is essential in this process. The underlying molecular mechanisms, however, remain unknown. We show that draper (drpr) and ced-6, which are essential for the clearance of apoptotic cells in C. elegans, function in the glial engulfment of larval axons during Drosophila metamorphosis. The drpr mutation and glia-specific knockdown of drpr and ced-6 by RNA interference suppress glial engulfment, resulting in the inhibition of axon pruning. drpr and ced-6 interact genetically in the glial action. Disruption of the microtubule cytoskeleton in the axons to be pruned occurs via ecdysone signaling, independent of glial engulfment. These findings suggest that glial cells engulf degenerating axons through drpr and ced-6. We propose that apoptotic cells and degenerating axons of living neurons are removed by a similar molecular mechanism.

Image-based modeling of the response of experimental 3D microstructures to mechanical loading

Abstract A three-dimensional (3D) spatial and crystallographic reconstruction of an austenitic steel microstructure was generated using optical microscopy, serial sectioning, and electron backscatter diffraction, and was incorporated into an image-based finite element model to simulate the mesoscale mechanical response of the real microstructure. The effects of crystallographic orientation and the interactions between applied loads, constraints, and microstructure are discussed. Advanced 3D visualization techniques are used to identify critical microstructural features at which the onset of plasticity, failure and fracture are predicted to occur.

Form and Function of on-off Amacrine Cells in the Amphibian Retina

ON-OFF amacrine cells were studied with whole cell recording techniques and intracellular staining methods using intact retina-eyecup preparations of the tiger salamander (Ambystoma tigrinum) and the mudpuppy (Necturus maculosus). Morphological characterization of these cells included three-dimensional reconstruction methods based on serial optical sections obtained with a confocal microscope. Some cells had their detailed morphology digitized with a computer-assisted tracing system and converted to compartmental models for computer simulations. The dendrites of ON-OFF amacrine cells have spines and numerous varicosities. Physiological recordings confirmed that ON-OFF amacrine cells generate both large- and small-amplitude impulses attributed, respectively, to somatic and dendritic generation sites. Using a multichannel model for impulse generation, computer simulations were carried out to evaluate how impulses are likely to propagate throughout these structures. We conclude that the ON-OFF amacrine cell is organized with multifocal dendritic impulse generating sites and that both dendritic and somatic impulse activity contribute to the functional repertoire of these interneurons: locally generated dendritic impulses can provide regional activation, while somatic impulse activity results in rapid activation of the entire dendritic tree.

Three-dimensional micro-PTV using deconvolution microscopy

A three-dimensional micro-particle tracking velocimetry (micro-PTV) scheme is presented using a single camera with deconvolution microscopy. This method devises tracking of the line-of-sight (z) flow vectors by correlating the diffraction pattern ring size variations with the defocusing distances of small particle locations. The working principle is based on optical serial sectioning microscopy, or equivalently deconvolution microscopy, that records images of an infinitesimally small particle, and generates a point-spread function of the three-dimensional diffraction patterns. A new image-processing algorithm has also been developed to digitally identify the center locations and measure the radii of the diffraction rings, which allows simultaneous tracking of all three-vector components. The developed PTV technique uses a 40×, 0.75 NA dry objective lens with 500-nm fluorescent seeding particles of SG=1.05, and successfully measures the fully three-dimensional fields flowing over a spherical obstacle snuggly fitted inside a 100 μm × 100 μm micro-channel. The volumetric measurement resolution of the present system is equivalent to a 5.16 μm × 5.16 μm × 5.16 μm cube, and the overall measurement uncertainty for single-point velocity vector detection is estimated to ±7.58%.

A protocol to analyse cellular dynamics during plant development

In vivo microscopy generates images that contain complex information on the dynamic behaviour of three-dimensional (3D) objects. As a result, adapted mathematical and computational tools are required to help in their interpretation. Ideally, a complete software chain to study the dynamics of a complex 3D object should include: (i) the acquisition, (ii) the preprocessing and (iii) segmentation of the images, followed by (iv) a reconstruction in time and space and (v) the final quantitative analysis. Here, we have developed such a protocol to study cell dynamics at the shoot apical meristem in Arabidopsis. The protocol uses serial optical sections made with the confocal microscope. It includes specially designed algorithms to automate the identification of cell lineage and to analyse the quantitative behaviour of the meristem surface.

Reconstruction and representation of caudal vasculature of zebrafish embryo from confocal scanning laser fluorescence microscopic images

Three-dimensional (3D) reconstruction from a series of sections is an important technique in medical imaging, particularly for visualization of blood vessels from angiography. Here, we present a framework for automatic segmentation and registration of different kind of blood vessels from 2-day-old zebrafish embryos. Series of optical sections were acquired from confocal microscopy with the blood vessels labeled by fluorescent microbeads ( 0.02 μ m ) injected into blood stream of 2-day-old zebrafish embryos. Blood vessels were extracted and their morphological parameters, including length and diameter, were calculated. At the same time, individual blood vessels were registered automatically. Vasculature was represented by attributed vessel represent graph (AVRG), which contained morphological data and connectivity of every blood vessel. Using AVRG to represent a vasculature made the comparison between vasculatures of different embryos more easy. Visualization, as well as quantification, of reconstructed 3D model of AVRG was presented in an interactive interface. The framework was implemented by Visual C++ as Windows-based program.

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.

Profile of bordered pit aspiration in Cryptomeria japonica using confocal laser scanning microscopy: pit aspiration and heartwood color

Nine trees of Cryptomeria japonica from six elite tree clones with a broad range of heartwood colors were selected. The profiles of pit aspiration percentage (ASP) of earlywood and latewood from pith to bark for green and air-dry conditions were determined to study the relationship between heartwood color and pit aspiration. Confocal laser scanning microscopy (CLSM) observations showed that the ASP of earlywood was low in sapwood and high in heartwood in the green condition. Pit aspiration increased in intermediate wood when compared with sapwood. On the other hand, latewood pits did not aspirate during heartwood formation. Comparing the air-dry condition with the green condition, sapwood pits aspirated during drying in both earlywood and latewood; however, there was no significant difference in pit aspiration of heartwood. There was no significant difference between samples with red and black heartwoods for ASP. The difference in ASP between individual trees was larger than that by heartwood color. The general advantage of CLSM over light microscopy is that serial optical sections along the Z axis can be obtained for any moisture condition, without the need for thin sectioning or embedding.

Temperature measurement for a nanoparticle suspension by detecting the Brownian motion using optical serial sectioning microscopy (OSSM)

A concept of nonintrusive thermometry is presented based on the correlation of the Brownian motion of suspended nanoparticles with the surrounding fluid temperature. Detection of fully three-dimensional Brownian motion is possible by the use of optical serial sectioning microscopy (OSSM). This technique measures optically diffracted particle images, the so-called point spread function (PSF), and determines the defocusing or line-of-sight location of the imaged particle measured from the focal plane. A dry objective lens (40×, 0.75 NA) is used to detect the diffraction patterns of 500 nm polystyrene fluorescent (505/515) nanoparticles suspended in water at a volume concentration of 4 × 10−6, for a range of temperatures from 5 to 70 °C. The measured mean square displacement (MSD) data (figure 8, table 1) agree fairly well with the well-known Einstein predictions. Differentials of 5.54%, 4.26% and 3.19% were found for the 1D, 2D and 3D cases, respectively. In summary, the line-of-sight Brownian motion detection using the OSSM technique is proposed in lieu of the more cumbersome two-dimensional Brownian motion tracking on the imaging plane as a potentially more effective tool to nonintrusively map the temperature fields for nanoparticle suspension fluids.

Tumor-induced mechanical hyperalgesia involves CGRP receptors and altered innervation and vascularization of DsRed2 fluorescent hindpaw tumors

Functional and anatomical relationships among primary afferent fibers, blood vessels, and cancers are poorly understood. However, recent evidence suggests that physical and biochemical interactions between these peripheral components are important to both tumor biology and cancer-associated pain. To determine the role of these peripheral components in a mouse model of cancer pain, we quantified the change in nerve and blood vessel density within a fibrosarcoma tumor mass using stereological analysis of serial confocal optical sections of immunostained hind paw. To this end we introduced the Discoma coral-derived red fluorescent protein (DsRed2) into the NCTC 2472 fibrosarcoma line using the Sleeping Beauty transposon methodology, thus providing a unique opportunity to visualize tumor–nerve–vessel associations in context with behavioral assessment of tumor-associated hyperalgesia. Tumors from hyperalgesic mice are more densely innervated with calcitonin gene related peptide (CGRP)-immunoreactive nerve fibers and less densely vascularized than tumors from non-hyperalgesic mice. As hyperalgesia increased from Day 5 to 12 post-implantation, the density of protein gene product 9.5 (PGP9.5)-immunoreactive nerves and CD31-immunoreactive blood vessels in tumors decreased, whereas CGRP-immunoreactive nerve density remained unchanged. Importantly, intra-tumor injection of a CGRP1 receptor antagonist (CGRP 8–37) partially blocked the tumor-associated mechanical hyperalgesia, indicating that local production of CGRP may contribute to tumor-induced nociception through a receptor-mediated process. The results describe for the first time the interaction among sensory nerves, blood vessels and tumor cells in otherwise healthy tissue, and our assessment supports the hypothesis that direct tumor cell-axon communication may underlie, at least in part, the occurrence of cancer pain.

Subcellular Localization of Disease-Associated Prion Protein in the Human Brain

Disease-associated prion protein (PrP(TSE)) deposits in distinct immunostaining patterns in the brain in Creutzfeldt-Jakob disease, including synaptic, extracellular, and cell-associated localizations. After having developed an appropriate pretreatment protocol to enhance immunostaining for PrP(TSE) without damaging epitopes of other antigens, we systematically evaluated co-localization patterns of distinct PrP(TSE) immunodeposits by confocal laser microscopy, including optical serial sectioning. As shown by quantification, the most prominent co-localization of PrP(TSE) is with synaptophysin, but PrP(TSE) may also co-deposit with connexin-32, a gap junction-related protein. Furthermore, neuronal cell bodies, dendrites, axons, astrocytes, and microglia harbor granular PrP(TSE) deposits. Highly aggregated deposits are focally ubiquitinated. We conclude that PrP(TSE) is not exclusively associated with chemical but also with electric synapses, axonal transport may be a relevant route of PrP(TSE) spread in the brain, and activated microglia and astrocytes may play a role in PrP(TSE) processing, degradation, or removal.

Characterization of powdered epidermal vaccine delivery with multiphoton microscopy

Multiphoton laser scanning microscopy (MPLSM) has been adapted to non-invasively characterize hand-held powdered epidermal vaccine delivery technology. A near infrared femtosecond pulsed laser, wavelength at approximately 920 nm, was used to evoke autofluorescence of endogenous fluorophores within ex vivo porcine and human skin. Consequently, sub cellular resolution three-dimensional images of stratum corneum and viable epidermal cells were acquired and utilized to observe the morphological deformation of these cells as a result of micro-particle penetration. Furthermore, the distributional pattern of micro-particles within the specific skin target volume was quantified by measuring the penetration depth as revealed by serial optical sections in the axial plane obtained with MPLSM. Additionally, endogenous fluorescence contrast images acquired at the supra-basal layer reveal cellular structures that may pertain to dendritic Langerhans cells of the epidermis. These results show that MPLSM has advantages over conventional histological approaches, since three-dimensional functional images with sub-cellular spatial resolution to depths beyond the epidermis can be acquired non-invasively. Accordingly, we propose that MPLSM is ideal for investigations of powdered epidermal vaccine delivery.

The Hippocampal Cholinergic Neurostimulating Peptide, the N-terminal Fragment of the Secreted Phosphatidylethanolamine-binding Protein, Possesses a New Biological Activity on Cardiac Physiology

Phosphatidylethanolamine-binding protein (PEBP), alternatively named Raf-1 kinase inhibitor protein, is the precursor of the hippocampal cholinergic neurostimulating peptide (HCNP) corresponding to its natural N-terminal fragment, previously described to be released by hippocampal neurons. PEBP is a soluble cytoplasmic protein, also associated with plasma and reticulum membranes of numerous cell types. In the present report, using biochemistry and cell biology techniques, we report for the first time the presence of PEBP in bovine chromaffin cell, a well described secretion model. We have examined its presence at the subcellular level and characterized this protein on both secretory granule membranes and intragranular matrix. In addition, its presence in bovine chromaffin cell and platelet exocytotic medium, as well as in serum, was reported showing that it is secreted. Like many other proteins that lack signal sequence, PEBP may be secreted through non-classic signal secretory mechanisms, which could be due to interactions with granule membrane lipids and lipid rafts. By two-dimensional liquid chromatography-tandem mass spectrometry, HCNP was detected among the intragranular matrix components. The observation that PEBP and HCNP were secreted with catecholamines into the circulation prompted us to investigate endocrine effects of this peptide on cardiovascular system. By using as bioassay an isolated and perfused frog (Rana esculenta) heart preparation, we show here that HCNP acts on the cardiac mechanical performance exerting a negative inotropism and counteracting the adrenergic stimulation of isoproterenol. All together, these data suggest that PEBP and HCNP might be considered as new endocrine factors involved in cardiac physiology.

Filamentous-actins in human hepatocarcinoma cells with CLSM.

To establish a method for optical sections of HepG2 human hepatoblastoma cells with confocal laser scanning microscope (CLSM) and to study the spatial structure of filamentous actin (F-actin) in HepG2 cells. HepG2 cells were stained with FITC-phalloidin that specifically binds F-actin, with propidium iodide (PI) to the nucleus, and scanned with a CLSM to generate optically sectioned images. A series of optical sections taken successively at different focal levels in steps of 0.7 microm were reconstructed with the CLSM reconstruction program. CLSM images showed that the FITC-stained F-actin was abundant microfilament bundles parallel or netted through the whole cell and its processes. Most F-actin microfilaments extended through the cell from one part toward the other or run through the process. Some microfilaments were attached to the plasma membrane, or formed a structural bridge connecting to the neighboring cells. A method for double labeling HepG2 human hepatoblastoma cells and CLSM imaging F-actin microfilaments and nuclei by image thin optical sections and spatial structure was developed. It provides a very useful way to study the spatial structure of F-actin.

Three dimensional analysis of histone methylation patterns in normal and tumor cell nuclei.

Histone modifications represent an important epigenetic mechanism for the organization of higher order chromatin structure and gene regulation. Methylation of position-specific lysine residues in the histone H3 and H4 amino termini has linked with the formation of constitutive and facultative heterochromatin as well as with specifically repressed single gene loci. Using an antibody, directed against dimethylated lysine 9 of histone H3 and several other lysine methylation sites, we visualized the nuclear distribution pattern of chromatin flagged by these methylated lysines in 3D preserved nuclei of normal and malignant cell types. Optical confocal serial sections were used for a quantitative evaluation. We demonstrate distinct differences of these histone methylation patterns among nuclei of different cell types after exit of the cell cycle. Changes in the pattern formation were also observed during the cell cycle. Our data suggest an important role of methylated histones in the reestablishment of higher order chromatin arrangements during telophase/early G1. Cell type specific histone methylation patterns are possibly casually involved in the formation of cell type specific heterochromatin compartments, composed of (peri)centromeric regions and chromosomal subregions from neighboring chromosomes territories, which contain silent genes.

Novel, posterior sensory organ in the trochophore larva of Phyllodoce maculata (Polychaeta).

A new posterior sensory organ (PSO), located at the dorsal midline of the hyposphere, is described by immunocytochemical detection of acetylated alpha tubulin and serotonin (5-HT) in a laser-scanning microscope, as well as three-dimensional reconstructions after optical serial sectioning in the trochophore larva of the polychaete Phyllodoce maculata (Phyllodocidae). The unpaired PSO consists of five bipolar sensory cells, two of them being 5-HT immunopositive, which send axons to the cerebral ganglion and prototroch nerve. The dendrites of these cells project to the surface and bear one cilium each. A single neuronal fibre from the apical sensory organ innervates the PSO.

Triple immunofluorolabeling with two rabbit polyclonal antibodies and a mouse monoclonal antibody allowing three-dimensional analysis of cotton wool plaques in Alzheimer disease.

We established a triple-labeling method with two rabbit polyclonal antibodies and a mouse monoclonal antibody and examined autopsied brain tissue with cotton wool plaques (CWPs). One of the polyclonal antibodies was so diluted (anti-Abeta42 or anti-Abeta40/1:30,000 or anti-von Willebrand factor/1:1000) that its visualization was possible only after amplification with the catalyzed reporter deposition (CARD) method. The other polyclonal antibody (anti-Abeta40 or anti-Abeta42/1:1000) was visualized with a fluorochrome conjugated to an anti-rabbit antibody that specifically visualized the latter polyclonal antibody because of its lower sensitivity. A monoclonal antibody, AT8, was superimposed to yield triple immunofluorolabeling. Serial optical sections with an interval of 0.3 micro m were reconstructed to allow three-dimensional (3D) observation of these three epitopes. Abeta40 was localized to core-like structures, mainly in layers I-III, and was sometimes in contact with the vascular wall, both without neuritic reactions. CWPs, present in layers I-VI, were labeled with anti-Abeta42 and were accompanied by neuritic reactions. These differences suggest that mechanisms of Abeta deposition and its relation to neuritic reactions or to blood vessels differ according to the lesion, even in the same microscopic field.

Three-dimensional capillary geometry in gut tissue.

Accurate characterization of capillary geometry is of the utmost importance for physiological tissue studies such as oxygen transport. We show that 3D microscopy can be used to measure tissue capillary geometry both in normal and disease states. We imaged fluorescently labeled gut mucosa capillary beds of three control rats and three rats 4 hours after i.p. injection of 9 mg/kg endotoxin. We used serial optical sectioning microscopy coupled with deconvolution to reconstruct 3D capillary geometry. Theoretical point spread functions accounting for depth into the specimen resulted in better reconstructions than experimentally measured point spread functions. We next derived the distribution of the shortest distances to the nearest capillary from all extravascular tissue voxels. In normal rats the shortest-distance distributions were remarkably constant despite widely varying capillary geometry. Furthermore, the mean of the shortest-distance distributions increased significantly for endotoxemic rats (4.8+/-0.4 microm) compared to controls (4.3+/-0.3 microm, P<0.05). Hence, serial sectioning microscopy provides an accurate venue for measuring physiologically relevant 3D capillary structure.