Confocal Image Stacks Research Articles

Use of Multiphoton Intravital Imaging and a Multiscale Computational Model of Thrombus Development to Study the Role of FVII in Thrombogenesis

Using 2-photon intravital microscopy we have generated high resolution, near real-time 3-dimensional images of a developing thrombus. Following Titanium - Sapphire laser-induced injuries in mouse mesenteric vessels, the developing thrombus was monitored by collecting stacks of confocal images through the developing thrombus. Data were collected in 3 channels for fluorescently labeled platelets, fibrinogen and 70,000 MW dextran. By including fluorescently labeled dextran in the blood we were able to monitor flow (plasma) and not labeled cells (leukocytes and erythrocytes) forming black silhouettes in the plasma. Since each 3-D reconstruction involves a series of scans, we were able to generate approximately 2–3 reconstructions per minute. Thus, the system sacrifices temporal resolution for high resolution structural information revealing the changing, heterogeneous sub-domain structure of the developing thrombus. The imaging system has been used to study the consequences of FVII-deficiency. Following injury, of the luminal surface of the vessel, a thin layer of platelets and fibrin accumulated at the injury site. Unlike injuries in wild-type mice where the thrombus continues to grow, the injuries in FVII deficient mice failed to grow as a result of frequent embolization from the developing structure. Interestingly, in a model of ferric chloride induced injury of the carotid artery, thrombi in FVII deficient mice form large structures capable of reducing flow although, unlike wild type mice, the FVII deficient animals fail to form stable occlusive clots. In parallel with the modified experimental vascular injury model, we have begun development of a computational model of thrombus development. The modeling framework consists of a stochastic and discrete Cellular Potts Model (CPM) to describe platelet and cellular interactions and continuous submodels to describe hydrodynamic and biochemical reactions. Our multiscale model includes the vessel wall, platelets (in resting and activated states), blood cells, coagulation reactions, fibrin formation, and hydrodynamic parameters as components. By comparing the in vivo experimental results with those of simulations varying the concentration of FVII we are able to refine and validate the computational model of thrombogenesis.

A Fluorescent Two-hybrid Assay for Direct Visualization of Protein Interactions in Living Cells

Genetic high throughput screens have yielded large sets of potential protein-protein interactions now to be verified and further investigated. Here we present a simple assay to directly visualize protein-protein interactions in single living cells. Using a modified lac repressor system, we tethered a fluorescent bait at a chromosomal lac operator array and assayed for co-localization of fluorescent prey fusion proteins. With this fluorescent two-hybrid assay we successfully investigated the interaction of proteins from different subcellular compartments including nucleus, cytoplasm, and mitochondria. In combination with an S phase marker we also studied the cell cycle dependence of protein-protein interactions. These results indicate that the fluorescent two-hybrid assay is a powerful tool to investigate protein-protein interactions within their cellular environment and to monitor the response to external stimuli in real time.

Clathrin-dependent Endocytosis Is Required for TrkB-dependent Akt-mediated Neuronal Protection and Dendritic Growth

Endocytosis of Trk (tropomyosin-related kinase) receptors is critical for neurotrophin signal transduction and biological functions. However, the mechanism governing endocytosis of TrkB (tropomyosin-related kinase B) and the specific contributions of TrkB endocytosis to downstream signaling are unknown. In this study, we report that blocking clathrin, dynamin, or AP2 in cultured neurons of the central nervous system inhibited brain-derived neurotrophic factor (BDNF)-induced activation of Akt but not ERK. Treating neurons with the clathrin inhibitor monodansylcadaverine or a peptide that blocks dynamin function specifically abrogated Akt pathway activation in response to BDNF but did not affect the response of other downstream effectors or the up-regulation of immediate early genes neuropeptide Y and activity-regulated cytoskeleton-associated protein. Similar effects were found in neurons expressing small interfering RNA to silence AP2 or a dominant negative form of dynamin that inhibits clathrin-mediated endocytosis. In PC12 cells, ERK but not Akt activation required TrkA endocytosis following stimulation with nerve growth factor, whereas the opposite was true when TrkA-expressing neurons were stimulated with nerve growth factor in the central nervous system. Thus, the specific effects of internalized Trk receptors probably depend on the presence of cell type-specific modulators of neurotrophin signaling and not on differences inherent to Trk receptors themselves. Endocytosis-dependent activation of Akt in neurons was found to be critical for BDNF-supported survival and dendrite outgrowth. Together, these results demonstrate the functional requirement of clathrin- and dynamin-dependent endocytosis in generating the full intracellular response of neurons to BDNF in the central nervous system.

A statistical approach for intensity loss compensation of confocal microscopy images

In this paper, a probabilistic technique for compensation of intensity loss in confocal microscopy images is presented. For single-colour-labelled specimen, confocal microscopy images are modelled as a mixture of two Gaussian probability distribution functions, one representing the background and another corresponding to the foreground. Images are segmented into foreground and background by applying Expectation Maximization algorithm to the mixture. Final intensity compensation is carried out by scaling and shifting the original intensities with the help of parameters estimated for the foreground. Since foreground is separated to calculate the compensation parameters, the method is effective even when image structure changes from frame to frame. As intensity decay function is not used, complexity associated with estimation of the intensity decay function parameters is eliminated. In addition, images can be compensated out of order, as only information from the reference image is required for the compensation of any image. These properties make our method an ideal tool for intensity compensation of confocal microscopy images that suffer intensity loss due to absorption/scattering of light as well as photobleaching and the image can change structure from optical/temporal section-to-section due to changes in the depth of specimen or due to a live specimen. The proposed method was tested with a number of confocal microscopy image stacks and results are presented to demonstrate the effectiveness of the method.

Quantitative Comparison of Intracellular Unpacking Kinetics of Polyplexes by a Model Constructed From Quantum Dot-FRET

A major challenge for non-viral gene delivery is gaining a mechanistic understanding of the rate-limiting steps. A critical barrier in polyplex-mediated gene delivery is the timely unpacking of polyplexes within the target cell to liberate DNA for efficient gene transfer. In this study, the component plasmid DNA and polymeric gene carrier were individually labeled with quantum dots (QDs) and Cy5 dyes, respectively, as a donor and acceptor pair for fluorescence resonance energy transfer (FRET). The high signal-to-noise ratio in QD-mediated FRET enabled sensitive detection of discrete changes in polyplex stability. The intracellular uptake and dissociation of polyplexes through QD-FRET was captured over time by confocal microscopy. From quantitative image-based analysis, distributions of released plasmid within the endo/lysosomal, cytosolic, and nuclear compartments formed the basis for constructing a three-compartment first-order kinetics model. Polyplex unpacking kinetics for chitosan, polyethylenimine, and polyphosphoramidate were compared and found to correlate well with transfection efficiencies. Thus, QD-FRET-enabled detection of polyplex stability combined with image-based quantification is a valuable method for studying mechanisms involved in polyplex unpacking and trafficking within live cells. We anticipate that this method will also aid the design of more efficient gene carriers.

Repeated stress alters dendritic spine morphology in the rat medial prefrontal cortex

Anatomical alterations in the medial prefrontal cortex (mPFC) are associated with hypothalamopituitary adrenal (HPA) axis dysregulation, altered stress hormone levels, and psychiatric symptoms of stress-related mental illnesses. Functional imaging studies reveal impairment and shrinkage of the mPFC in such conditions, and these findings are paralleled by experimental studies showing dendritic retraction and spine loss following repeated stress in rodents. Here we extend this characterization to how repeated stress affects dendritic spine morphology in mPFC through the utilization of an automated approach that rapidly digitizes, reconstructs three dimensionally, and calculates geometric features of neurons. Rats were perfused after being subjected to 3 weeks of daily restraint stress (6 hours/day), and intracellular injections of Lucifer Yellow were made in layer II/III pyramidal neurons in the dorsal mPFC. To reveal spines in all angles of orientation, deconvolved high-resolution confocal laser scanning microscopy image stacks of dendritic segments were reconstructed and analyzed for spine volume, surface area, and length using a Rayburst-based automated approach (8,091 and 8,987 spines for control and stress, respectively). We found that repeated stress results in an overall decrease in mean dendritic spine volume and surface area, which was most pronounced in the distal portion of apical dendritic fields. Moreover, we observed an overall shift in the population of spines, manifested by a reduction in large spines and an increase in small spines. These results suggest a failure of spines to mature and stabilize following repeated stress and are likely to have major repercussions on function, receptor expression, and synaptic efficacy.

Correlative electron and confocal microscopy assessment of synapse localization in the central nervous system of an insect

Excellent methods exist to analyze sub-neuronal structures, such as synapses, at nanometer resolution with electron microscopy. However, due to methodological constraints, electron microscopy is feasible only for small volumes of fixed tissue. By contrast, confocal or two-photon laser scanning microscopy is well suited to obtain neuronal structures from large volumes of living or fixed tissue at sub-micrometer resolution. Therefore, a gap exists when analyzing synaptic organization of neuropils, or the distribution of synapses throughout the dendritic trees of individual neurons, and it would be advantageous to use confocal microscopy to investigate the synaptic organization of central neuropils. This study uses correlative electron and confocal microscopy from the same tissue sections to test whether synapsin I-immunopositive puncta can be analyzed at the light microscopy level to estimate the distributions of synaptic sites within central motor neuropils and along reconstructed dendritic surfaces in an insect ventral nerve cord. It demonstrates that every type 1 synaptic terminal can be detected as a distinct punctum by synapsin I-immunolabeling and confocal microscopy. Furthermore, it provides data indicating that co-localization analysis from confocal image stacks as recently published provides a good estimate for quantifying the distribution patterns of input synapses through dendritic trees.

Computer-Aided Classification of Cells in Complex Brain Tissue from 5-Channel 3-D Confocal Datasets

Abstract Spectral Unmixing and Image Segmentation Cells Classification Experimental Results and Validation R1R2FundamentalScienceFundamentalValidatingTestBEDsTestBEDsL1L1L2L3R3S1 S2 S3 S4 S5 Bio-Med Enviro-Civil The cellular organization of brain tissue is truly complex. This work presents a computational method to identify the principal cell types in three-dimensional (3-D) confocal image stacks with multiple fluorescent channels. The cells are classified into four major classes (Neurons, Microglia, Astrocytes and Endothelials) by using a two-step classifier that applies fuzzy c-means clustering followed by Support Vector Machines (SVM). The resulting classification results were validated against a human expert, and the accuracy of the classifier was %95.5 in the correctly segmented nuclei. Features Extraction SpectralUnmixingNisslChannelIba-1ChannelTraceProcessesGFAPChannelTraceProcessesConvergenceAnalysisEBAChannelSurfaceSegmentationCyQuantChannelNuclearSegmentationInspect &EditSegmentationOutput

Analysis of Kupffer's vesicle in zebrafish embryos using a cave automated virtual environment

Previous studies show that cilia in Kupffer's vesicle (KV) generate a counterclockwise flow of fluid and provide convincing evidence that this flow regulates left/right asymmetry. We hypothesized that the distribution of cilia in KV leads to this directional flow. However, there are limitations in determining the localization of structures when viewing a three-dimensional (3-D) image on a 2-D computer screen. We analyzed the distribution of KV cilia in the Cave, an immersive virtual environment that displays stacks of confocal images in 3-D. We found 80% of the cilia are located on the dorsal surface and 20% were located on the ventral surface of the vesicle. We confirmed the ventral location of some cilia by electron microscopy. There is an asymmetrical distribution of cilia on the dorsal surface, with the anterior one third containing 50% and the posterior one third containing 20% of the cilia. This dorsal-anterior patch could explain the directionality of the flow, and could drive local differences in flow rate.

Automatic thresholding of three‐dimensional microvascular structures from confocal microscopy images

We have combined confocal microscopy, image processing, and optimization techniques to obtain automated, accurate volumetric measurements of microvasculature. Initially, we made tissue phantoms containing 15-microm FocalCheck microspheres suspended in type I collagen. Using these phantoms we obtained a stack of confocal images and examined the accuracy of various thresholding schemes. Thresholding algorithms from the literature that utilize a unimodal histogram, a bimodal histogram, or an intensity and edge-based algorithm all significantly overestimated the volume of foreground structures in the image stack. Instead, we developed a heuristic technique to automatically determine good-quality threshold values based on the depth, intensity, and (optionally) gradient of each voxel. This method analyzed intensity and gradient threshold methods for each individual image stack, taking into account the intensity attenuation that is seen in deeper images of the stack. Finally, we generated a microvascular construct comprised of rat fat microvessel fragments embedded in collagen I gels and obtained stacks of confocal images. Using our new thresholding scheme we were able to obtain automatic volume measurements of growing microvessel fragments.

Neurons of the ascidian larval nervous system in Ciona intestinalis: II. Peripheral nervous system

The peripheral nervous system of the ascidian tadpole larva comprises a distributed population of isolated receptor neurons, most of unproved function, organized along the trunk or tail epithelium. Previous reports using immunocytochemical methods failed to resolve the detailed morphology of the neurons and their axon pathways. Precleavage embryos of Ciona intestinalis transfected with the promoter of the neuron-specific synaptotagmin gene fused to a green fluorescent protein (GFP) gene yielded clearly labelled GFP profiles. These we examined in confocal image stacks of 31 larvae. Anchor cells, at least eight in each adhesive apical papilla, contribute axons to the papillar nerves that terminate in the sensory vesicle of the central nervous system. Two nerve bundles projected from each papilla, suggesting that at least two subpopulations of papillar neurons exist. Each bundle fasciculated with axons of the rostral trunk epidermal neurons (RTEN) in a stereotyped pattern. The RTEN had a hitherto unreported elaborate arbor of sensory dendrites within the tunic, suggesting that each has an extended sensorial field. Two subpopulations of apical trunk epidermal neurons (ATEN), anterior and posterior, were distinguished. As with the RTEN, these neurons extended dendritic arbors into the tunic. Two additional types of tail neuron, the caudal epidermal neurons (dorsal and ventral) as well as a novel bipolar interneuron, were identified. These identified neuron types are the substrate for the ascidian larva's entire peripheral sensory input, important during larval swimming and settlement.

Neurons of the ascidian larval nervous system in Ciona intestinalis: I. Central nervous system

The tadpole larva of ascidians, basal living relatives of vertebrates, has a chordate body plan. The CNS has many homologies with that of vertebrates yet only about 100 neurons. These few, possibly fixed in number and composition, nevertheless govern a diverse repertoire of behaviors. To elucidate the circuits of the CNS first requires that we recognize each neuron type, for which we used electroporation to transfect precleavage embryos with a plasmid containing green fluorescent protein (GFP) driven by the promoter of the synaptotagmin gene. Hatched larvae were fixed and GFP 3-D reconstructions of confocal image stacks compiled into images of 31 whole or partial larvae, either with many GFP-labelled neurons or with few, each clearly visible. Neuron counts in the sensory vesicle (SV) and visceral ganglion (VG) indicated that between 75% (SV) and 69% (VG) of previously reported numbers of neurons were transfected. Based on their position, shape, and projections, the following neurons were identified in the SV: a prominent eminens neuron, possibly with direct input from papillar neurons, a large ventroposterior interneuron, photoreceptors of the ocellus, and putative antenna cells of the otolith. In the VG, we identified at least four subtypes of motor neuron, including an ovoid cell that may innervate distal tail muscle cells and contrapelo cells with ascending projections, unique among VG neurons. The caudal nerve cord contained the first reported neurons, the somata of planate neurons. These neurons are the first identified types, and will be used to construct a map of the nervous system for this model basal chordate.

Activation-dependent trafficking of NTPDase2 in Chinese hamster ovary cells

Membrane-bound NTPDase2 is a member of the ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) enzyme family involved in the regulation of P2 receptor signaling. NTPDase2 has broad substrate specificity for extracellular nucleotides, but hydrolyses nucleoside 5′-triphosphates with high preference over nucleoside 5′-diphosphates. In this study, we have sought to determine how enzyme substrates acting on P2 receptors affect intracellular NTPDase2 trafficking. To achieve this, Chinese hamster ovary (CHO) cells were transiently transfected with rat-specific NTPDase2 cDNA tagged with green fluorescent protein (GFP), to allow direct visualisation of subcellular localisation and trafficking of NTPDase2. Cells were superfused with NTPDase2 substrates (ATP and UTP) and synthetic nucleotide analogues (ATPγS and ADPβS), and confocal image stacks were acquired at regular time intervals. NTPDase2 incorporation into the plasma membrane was determined by comparative analysis of fluorescence intensity in the cytosolic and membrane compartments. GFP-tagged NTPDase2 was fully functional and ATP and ATPγS induced membrane incorporation of GFP-NTPDase2 from putative intracellular stores, whilst UTP and ADPβS were ineffective. The increased ATP hydrolysis rate correlated with increased NTPDase2 trafficking to the plasma membrane. ATP-induced NTPDase2 trafficking was mediated by activation of endogenous P2X receptors involving Ca2+ entry rather than by P2Y receptor-induced release of Ca2+ from intracellular stores. Our results suggest that P2X receptor activation stimulates insertion of latent NTPDase2 into the plasma membrane. The increase in surface-located NTPDase2 may reflect a regulatory mechanism counteracting excessive stimulation and desensitisation of P2 receptors.

Real-time reflectance confocal microscopy: comparison of two-dimensional images and three-dimensional image stacks for detection of cervical precancer

Confocal microscopy can provide real-time, 2-D and 3-D images of the cellular morphology and tissue architecture features that pathologists use to detect precancerous lesions without the need for tissue removal, sectioning, and staining. The utility of 3-D confocal image stacks of epithelial tissue for detecting dysplasia has not yet been explored. We aim to extract morphometry and tissue architecture information from 2-D confocal reflectance images and 3-D image stacks from fresh, unstained cervical biopsies and compare their potential for detecting dysplasia. Nine biopsies are obtained from eight patients; confocal images are acquired pre- and postacetic acid at multiple epithelial depths in 1.5 mum-intervals. Postacetic acid images are processed to segment cell nuclei; after segmentation, 2-D images taken at 50 mum below the tissue surface, and the entire 3-D image stacks are processed to extract morphological and architectural features. Data are analyzed to determine which features gave the best separation between normal and high-grade cervical precancer. Most significant differences are obtained from parameters extracted from the 3-D image stacks. However, in all cases where the 2-D features were multiplicatively scaled by the depth of acquisition divided by the epithelial thickness or scaled by the scattering coefficient, the significance level is equal to or greater than the comparable feature extracted from the 3-D image stacks. A linear discriminant function previously developed to separate 19 samples of normal tissue and high-grade cervical precancer based on the nuclear-to-cytoplasm (N/C) ratio and epithelial scattering coefficient is prospectively applied to the nine biopsies examined to determine the accuracy with which it could separate normal tissue from cervical intra epithelial neoplasia (CIN) 23. For the entire data set of 28 biopsies, a sensitivity and specificity of 100% is produced using this discriminant function; the scattering coefficient provides more discriminative capacity than the N/C ratio. The success of the scaled 2-D image features has important implications for using confocal microscopy to detect precancer in the clinic. Acquisition of the epithelial thickness or scattering coefficient requires less time than 3-D image sets and little additional effort is required to gain the added information compared to 2-D images alone.

Phosphorylation-Dependent Control of Pc2 SUMO E3 Ligase Activity by Its Substrate Protein HIPK2

Sumoylation serves to control key cellular functions, but the regulation of SUMO E3 ligase activity is largely unknown. Here we show that the polycomb group protein Pc2 binds to and colocalizes with homeodomain interacting protein kinase 2 (HIPK2) and serves as a SUMO E3 ligase for this kinase. DNA damage-induced HIPK2 directly phosphorylates Pc2 at multiple sites, which in turn controls Pc2 sumoylation and intranuclear localization. Inducible phosphorylation of Pc2 at threonine 495 is required for its ability to increase HIPK2 sumoylation in response to DNA damage, thereby establishing an autoregulatory feedback loop between a SUMO substrate and its cognate E3 ligase. Sumoylation enhances the ability of HIPK2 to mediate transcriptional repression, thus providing a mechanistic link for DNA damage-induced transcriptional silencing.

Improving the Repeatability of Topographic Height Measurements in Confocal Scanning Laser Imaging Using Maximum-Likelihood Deconvolution

To evaluate maximum likelihood (ML) blind deconvolution as a technique for improving the repeatability of topographic height measurements obtained from scanning laser tomography (Heidelberg Retinal Tomograph [HRT]; Heidelberg Engineering, Heidelberg, Germany). ML blind deconvolution is an image-processing technique that estimates the original scene from a degraded image. This technique has been used in confocal scanning laser microscopy to remove "out-of-focus" haze in three-dimensional confocal image stacks. ML blind deconvolution requires no prior estimation of the point-spread function (PSF), as opposed to classic linear deconvolution methods. Instead, the algorithm estimates an initial PSF based on the optical setup of the confocal scanning device and optics of the eye and iteratively proceeds to a solution. The improvement in repeatability of height measurements from mean topography images within scan (intrascan) and between scans (interscan) afforded by ML deconvolution was evaluated in a test-retest series of HRT images from 40 ocular hypertensive and glaucomatous patients with varying degrees of media opacity. There was an improvement in intrascan repeatability in 38 out of the 40 mean topography images (median improvement 2.5 microm, inter-quartile range 2.19, P < 0.001), and an improvement in interscan repeatability in 33 of the 40 mean topographies (median improvement, 1.0 microm, interquartile range 3.49, P < 0.001). There was a positive association between the magnitude of the improvement in repeatability and the level of mean pixel height standard deviation (MPHSD), intrascan (P = 0.004) and interscan (P = 0.002). ML blind deconvolution algorithm improves the repeatability of topographic height measurements from the HRT. This improvement was greater in patients with poorer quality images.

Activity-Independent Prespecification of Synaptic Partners in the Visual Map of Drosophila

Specifying synaptic partners and regulating synaptic numbers are at least partly activity-dependent processes during visual map formation in all systems investigated to date . In Drosophila, six photoreceptors that view the same point in visual space have to be sorted into synaptic modules called cartridges in order to form a visuotopically correct map . Synapse numbers per photoreceptor terminal and cartridge are both precisely regulated . However, it is unknown whether an activity-dependent mechanism or a genetically encoded developmental program regulates synapse numbers. We performed a large-scale quantitative ultrastructural analysis of photoreceptor synapses in mutants affecting the generation of electrical potentials (norpA, trp;trpl), neurotransmitter release (hdc, syt), vesicle endocytosis (synj), the trafficking of specific guidance molecules during photoreceptor targeting (sec15), a specific guidance receptor required for visual map formation (Dlar), and 57 other novel synaptic mutants affecting 43 genes. Remarkably, in all these mutants, individual photoreceptors form the correct number of synapses per presynaptic terminal independently of cartridge composition. Hence, our data show that each photoreceptor forms a precise and constant number of afferent synapses independently of neuronal activity and partner accuracy. Our data suggest cell-autonomous control of synapse numbers as part of a developmental program of activity-independent steps that lead to a "hard-wired" visual map in the fly brain.

Tropoelastin Massively Associates during Coacervation To Form Quantized Protein Spheres

Tropoelastin, the precursor of elastin, undergoes a rapid monomer to multimer association in an inverse temperature transition. This association culminates in the rapid formation of stable, optically distinct droplets of tropoelastin. Light scattering and microscope measurements reveal that these droplets are 2-6 microm in diameter. Scanning electron microscopy confirms that the droplets are spherical. Three-dimensional confocal image stacks based on the autofluorescence of tropoelastin reveal that droplets are loaded with hydrated tropoelastin. Droplets are viable intermediates in synthetic elastin macroassembly. Dense clusters of aggregated droplets and partially formed fibers develop when droplets are incubated in the presence of a lysyl oxidase. Lysine-reacting chemical and enzyme-assisted cross-linking conditions generate cross-linked beads due to interactions between multiple, surface-exposed lysine epsilon-amino groups. Droplets represent an efficient mechanism for the bolus delivery during elastogenesis of quantized packages of preaccreted tropoelastin.

Nitric oxide-induced mitochondrial fission is regulated by dynamin-related GTPases in neurons

Mitochondria are present as tubular organelles in neuronal projections. Here, we report that mitochondria undergo profound fission in response to nitric oxide (NO) in cortical neurons of primary cultures. Mitochondrial fission by NO occurs long before neurite injury and neuronal cell death. Furthermore, fission is accompanied by ultrastructural damage of mitochondria, autophagy, ATP decline and generation of free radicals. Fission is occasionally asymmetric and can be reversible. Strikingly, mitochondrial fission is also an early event in ischemic stroke in vivo. Mitofusin 1 (Mfn1) or dominant-negative Dynamin related protein 1 (Drp1(K38A)) inhibits mitochondrial fission induced by NO, rotenone and Amyloid-beta peptide. Conversely, overexpression of Drp1 or Fis1 elicits fission and increases neuronal loss. Importantly, NO-induced neuronal cell death was mitigated by Mfn1 and Drp1(K38A). Thus, persistent mitochondrial fission may play a causal role in NO-mediated neurotoxicity.

Three-dimensional mitochondrial arrangement in ventricular myocytes: from chaos to order

We have developed a novel method to quantitatively analyze mitochondrial positioning in three dimensions. Using this method, we compared the relative positioning of mitochondria in adult rat and rainbow trout (Oncorhynchus mykiss) ventricular myocytes. Energetic data suggest that trout, in contrast to the rat, have two subpopulations of mitochondria in their cardiomyocytes. Therefore, we speculated whether trout cardiomyocytes exhibit two types of mitochondrial patterns. Stacks of confocal images of mitochondria were acquired in live cardiomyocytes. The images were processed and mitochondrial centers were detected automatically. The mitochondrial arrangement was analyzed by calculating the three-dimensional probability density and distribution functions describing the distances between neighboring mitochondrial centers. In the rat (8 cells with a total of 7,546 mitochondrial centers), intermyofibrillar mitochondria are highly ordered and arranged in parallel strands. These strands are separated by approximately 1.8 mum and can be found in any transversal direction relative to each other. Neighboring strands exhibit the same mitochondrial periodicity. In contrast to the rat, trout ventricular myocytes (22 cells; 5,528 mitochondrial centers) exhibit a relatively chaotic mitochondrial pattern. Neighboring mitochondria can be found in any direction relative to each other. Thus, two potential subpopulations of mitochondria in trout are not distinguishable by their pattern. The developed method required minor interaction in the filtering of the mitochondrial centers. It is therefore a practical approach to describe intracellular organization and may also be used for analysis of time-dependent organizational changes. The obtained quantitative description of mitochondrial organization is a requisite for accurate mathematical analysis of mitochondrial systems biology.