High-resolution Confocal Imaging Research Articles

Exocyst-Dependent Membrane Addition Is Required for Anaphase Cell Elongation and Cytokinesis in Drosophila.

Mitotic and cytokinetic processes harness cell machinery to drive chromosomal segregation and the physical separation of dividing cells. Here, we investigate the functional requirements for exocyst complex function during cell division in vivo, and demonstrate a common mechanism that directs anaphase cell elongation and cleavage furrow progression during cell division. We show that onion rings (onr) and funnel cakes (fun) encode the Drosophila homologs of the Exo84 and Sec8 exocyst subunits, respectively. In onr and fun mutant cells, contractile ring proteins are recruited to the equatorial region of dividing spermatocytes. However, cytokinesis is disrupted early in furrow ingression, leading to cytokinesis failure. We use high temporal and spatial resolution confocal imaging with automated computational analysis to quantitatively compare wild-type versus onr and fun mutant cells. These results demonstrate that anaphase cell elongation is grossly disrupted in cells that are compromised in exocyst complex function. Additionally, we observe that the increase in cell surface area in wild type peaks a few minutes into cytokinesis, and that onr and fun mutant cells have a greatly reduced rate of surface area growth specifically during cell division. Analysis by transmission electron microscopy reveals a massive build-up of cytoplasmic astral membrane and loss of normal Golgi architecture in onr and fun spermatocytes, suggesting that exocyst complex is required for proper vesicular trafficking through these compartments. Moreover, recruitment of the small GTPase Rab11 and the PITP Giotto to the cleavage site depends on wild-type function of the exocyst subunits Exo84 and Sec8. Finally, we show that the exocyst subunit Sec5 coimmunoprecipitates with Rab11. Our results are consistent with the exocyst complex mediating an essential, coordinated increase in cell surface area that potentiates anaphase cell elongation and cleavage furrow ingression.

Sample preparation for high-resolution 3D confocal imaging of mouse skeletal tissue.

High-resolution confocal imaging is a vital tool for analyzing the 3D architecture and detailed spatial distribution of cells in situ. However, imaging of skeletal tissue has remained technically challenging because of its calcified nature. Here we describe a protocol that allows high-resolution imaging of skeletal tissue with preservation of cellular morphology and tissue architecture. The procedure involves tissue fixation, decalcification and cryosectioning of the mouse skeletal tissue to generate thick sections. The thick sections generated by this procedure are not only compatible with the analysis of genetically expressed fluorescent proteins but they also preserve antigenicity, thus enabling diverse combinations of antibody labeling. Further, this procedure also permits other fluorescence techniques such as TUNEL and ethynyl deoxyuridine (EdU) incorporation assays. Images resulting from the confocal imaging can be assessed qualitatively and quantitatively to analyze various parameters such as distribution and interrelationships of cell types. The technique is straightforward and robust, highly reproducible and can be completed in ∼11 d.

A Systems Approach Identifies Essential FOXO3 Functions at Key Steps of Terminal Erythropoiesis.

Circulating red blood cells (RBCs) are essential for tissue oxygenation and homeostasis. Defective terminal erythropoiesis contributes to decreased generation of RBCs in many disorders. Specifically, ineffective nuclear expulsion (enucleation) during terminal maturation is an obstacle to therapeutic RBC production in vitro. To obtain mechanistic insights into terminal erythropoiesis we focused on FOXO3, a transcription factor implicated in erythroid disorders. Using an integrated computational and experimental systems biology approach, we show that FOXO3 is essential for the correct temporal gene expression during terminal erythropoiesis. We demonstrate that the FOXO3-dependent genetic network has critical physiological functions at key steps of terminal erythropoiesis including enucleation and mitochondrial clearance processes. FOXO3 loss deregulated transcription of genes implicated in cell polarity, nucleosome assembly and DNA packaging-related processes and compromised erythroid enucleation. Using high-resolution confocal microscopy and imaging flow cytometry we show that cell polarization is impaired leading to multilobulated Foxo3 -/- erythroblasts defective in nuclear expulsion. Ectopic FOXO3 expression rescued Foxo3 -/- erythroblast enucleation-related gene transcription, enucleation defects and terminal maturation. Remarkably, FOXO3 ectopic expression increased wild type erythroblast maturation and enucleation suggesting that enhancing FOXO3 activity may improve RBCs production. Altogether these studies uncover FOXO3 as a novel regulator of erythroblast enucleation and terminal maturation suggesting FOXO3 modulation might be therapeutic in disorders with defective erythroid maturation.

High-resolution confocal imaging of wall ingrowth deposition in plant transfer cells: Semi-quantitative analysis of phloem parenchyma transfer cell development in leaf minor veins of Arabidopsis.

BackgroundTransfer cells (TCs) are trans-differentiated versions of existing cell types designed to facilitate enhanced membrane transport of nutrients at symplasmic/apoplasmic interfaces. This transport capacity is conferred by intricate wall ingrowths deposited secondarily on the inner face of the primary cell wall, hence promoting the potential trans-membrane flux of solutes and consequently assigning TCs as having key roles in plant growth and productivity. However, TCs are typically positioned deep within tissues and have been studied mostly by electron microscopy.Recent advances in fluorophore labelling of plant cell walls using a modified pseudo-Schiff-propidium iodide (mPS-PI) staining procedure in combination with high-resolution confocal microscopy have allowed visualization of cellular details of individual tissue layers in whole mounts, hence enabling study of tissue and cellular architecture without the need for tissue sectioning. Here we apply a simplified version of the mPS-PI procedure for confocal imaging of cellulose-enriched wall ingrowths in vascular TCs at the whole tissue level.ResultsThe simplified mPS-PI staining procedure produced high-resolution three-dimensional images of individual cell types in vascular bundles and, importantly, wall ingrowths in phloem parenchyma (PP) TCs in minor veins of Arabidopsis leaves and companion cell TCs in pea. More efficient staining of tissues was obtained by replacing complex clearing procedures with a simple post-fixation bleaching step. We used this modified procedure to survey the presence of PP TCs in other tissues of Arabidopsis including cotyledons, cauline leaves and sepals. This high-resolution imaging enabled us to classify different stages of wall ingrowth development in Arabidopsis leaves, hence enabling semi-quantitative assessment of the extent of wall ingrowth deposition in PP TCs at the whole leaf level. Finally, we conducted a defoliation experiment as an example of using this approach to statistically analyze responses of PP TC development to leaf ablation.ConclusionsUse of a modified mPS-PI staining technique resulted in high-resolution confocal imaging of polarized wall ingrowth deposition in TCs. This technique can be used in place of conventional electron microscopy and opens new possibilities to study mechanisms determining polarized deposition of wall ingrowths and use reverse genetics to identify regulatory genes controlling TC trans-differentiation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0483-8) contains supplementary material, which is available to authorized users.

A thin layer angiogenesis assay: a modified basement matrix assay for assessment of endothelial cell differentiation.

BackgroundBasement matrices such as Matrigel™ and Geltrex™ are used in a variety of cell culture assays of anchorage-dependent differentiation including endothelial cell tube formation assays. The volumes of matrix recommended for these assays (approximately 150 μl/cm2) are costly, limit working distances for microscopy, and require cell detachment for subsequent molecular analysis. Here we describe the development and validation of a thin-layer angiogenesis (TLA) assay for assessing the angiogenic potential of endothelial cells that overcomes these limitations.ResultsGeltrex™ basement matrix at 5 μl/cm2 in 24-well (10 μl) or 96-well (2 μl) plates supports endothelial cell differentiation into tube-like structures in a comparable manner to the standard larger volumes of matrix. Since working distances are reduced, high-resolution single cell microscopy, including DIC and confocal imaging, can be used readily. Using MitoTracker dye we now demonstrate, for the first time, live mitochondrial dynamics and visualise the 3-dimensional network of mitochondria present in differentiated endothelial cells. Using a standard commercial total RNA extraction kit (Qiagen) we also show direct RNA extraction and RT-qPCR from differentiated endothelial cells without the need to initially detach cells from their supporting matrix.ConclusionsWe present here a new thin-layer assay (TLA) for measuring the anchorage-dependent differentiation of endothelial cells into tube-like structures which retains all the characteristics of the traditional approach but with the added benefit of a greatly lowered cost and better compatibility with other techniques, including RT-qPCR and high-resolution microscopy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12860-014-0041-5) contains supplementary material, which is available to authorized users.

Immunofluorescence detection of callose deposition around plasmodesmata sites.

Accumulation of callose (β-1,3 glucans) at the plasmodesmata (PD) neck region dynamically regulates symplastic intercellular transport. Here we describe a 2-3-day immuno-labelling protocol to determine callose levels in the cell wall region at PD. The method relies on exposure of internal cell walls by hand-sectioning of the sample and digestion of the cell wall with enzymes in order to improve antibody penetration to deep tissue layers. By using this protocol, combined with high-resolution confocal imaging, we successfully detected PD-associated callose in Arabidopsis root apical meristem, vascular tissue, and developing lateral root primordia.

Successful FCS experiment in nonstandard conditions.

Fluorescence correlation spectroscopy (FCS) is frequently used to measure the self-diffusion coefficient of fluorescently labeled probes in solutions, complex media, and living cells. In a standard experiment water immersion objectives and window thickness in the range of 0.13-0.19 mm are used. We show that successful FCS measurements can be performed using samples of different refractive index placed in cells having windows of different thickness, even much thicker than nominally allowed. Different water, oil, and silicon oil immersion as well as long working distance dry objectives, equipped with the correction collar, were tested and compared. We demonstrate that the requirements for FCS experiments are less stringent than those for high resolution confocal imaging and reliable relative FCS measurements can be performed even beyond the compensation range of the objectives. All these features open new possibilities for construction of custom-made high temperature and high pressure cells for FCS.

In vivo immunostaining of hemocyte compartments in Drosophila for live imaging.

In recent years, Drosophila melanogaster has become an attractive model organism in which to study the structure and development of the cellular immune components. The emergence of immunological markers greatly accelerated the identification of the immune cells (hemocytes), while the creation of genetic reporter constructs allowed unique insight into the structural organization of hematopoietic tissues. However, investigation of the hemocyte compartments by the means of immunological markers requires dissection and fixation, which regularly disrupt the delicate structure and hamper the microanatomical characterization. Moreover, the investigation of transgenic reporters alone can be misleading as their expression often differs from the native expression pattern of their respective genes. We describe here a method that combines the reporter constructs and the immunological tools in live imaging, thereby allowing use of the array of available immunological markers while retaining the structural integrity of the hematopoietic compartments. The procedure allows the reversible immobilization of Drosophila larvae for high-resolution confocal imaging and the time-lapse video analysis of in vivo reporters. When combined with our antibody injection-based in situ immunostaining assay, the resulting double labeling of the hemocyte compartments can provide new information on the microanatomy and functional properties of the hematopoietic tissues in an intact state. Although this method was developed to study the immune system of Drosophila melanogaster, we anticipate that such a combination of genetic and immunological markers could become a versatile technique for in vivo studies in other biological systems too.

Ex vivo culture of mouse embryonic skin and live-imaging of melanoblast migration.

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles(1,2). Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture(3-6). Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue(3,6). High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture(6). By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6 cultures in parallel.

Fluorescent labeling of dendritic spines in cell cultures with the carbocyanine dye "DiI".

Analyzing cell morphology is a key component to understand neuronal function. Several staining techniques have been developed to facilitate the morphological analysis of neurons, including the use of fluorescent markers, such as DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate). DiI is a carbocyanine membrane dye that exhibits enhanced fluorescence upon insertion of its lipophilic hydrocarbon chains into the lipid membrane of cells. The high photostability and prominent fluorescence of the dye serves as an effective means of illuminating cellular architecture in individual neurons, including detailed dendritic arborizations and spines in cell culture and tissue sections. Here, we specifically optimized a simple and reliable method to fluorescently label and visualize dissociated hippocampal neurons using DiI and high-resolution confocal microscopic imaging. With high efficacy, this method accurately labels neuronal and synaptic morphology to permit quantitative analysis of dendritic spines. Accurate imaging techniques of these fine neuronal specializations are vital to the study of their morphology and can help delineate structure-function relationships in the central nervous system.

The Technology Development Studio of the MPI-CBG: An Open Access Cell-Based Screening Facility

In the past decade, academic screening centers have been created in many universities worldwide. Most of these screening centers are organized as core facilities that accept projects from both within their organization and from external users in order to maximize staff and instrument usage. The Max Planck Institute of Molecular Cell Biology and Genetics in Dresden, Germany, also created such a screening facility named the Technology Development Studio (TDS). The mission of the facility is to provide cell based screening services focused on high resolution confocal imaging. The acquisition of high resolution images allows the mathematical description of cells with image analysis to a high degree of precision. This precision in turn allows classifying phenotypes accurately and compare different cellular treatments to discover underlying mechanisms.

Well-plate mechanical confinement platform for studies of mechanical mutagenesis

Limited space for cell division, perhaps similar to the compressed microenvironment of a growing tumor, has been shown to induce phenotypic and karyotypic changes to a cell during mitosis. To expand understanding of this missegregation of chromosomes in aberrant multi-daughter or asymmetric cell divisions, we present a simple technique for subjecting mammalian cells to adjustable levels of confinement which allows subsequent interrogation of intracellular molecular components using high resolution confocal imaging. PDMS micropatterned confinement structures of subcellular height with neighboring taller media reservoir channels were secured on top of confluent cells with a custom compression well-plate system. The system improved ease of use over previous devices since confined cells could be initially grown on glass coverslips in a 12-well plate, and subsequently be imaged by high resolution confocal imaging, or during compression by live cell imaging. Live cell imaging showed a significant increase in abnormal divisions of confined cells across three different cell lines (HeLa, A375, and A549). Immunofluoresecence stains revealed a significant increase in cell diameter and chromosome area of confined cells, but no significant increase in centrosome-centromere distance upon division when compared to unconfined cells. The developed system could open up studies more broadly on confinement effects on mitotic processes, and increase the throughput of such studies.

Synaptic pattern of nicotinic acetylcholine receptor α7 and β2 subunits on the direction-selective retinal ganglion cells in the postnatal mouse retina

Direction-selective retinal ganglion cells (DS RGCs) respond strongly to a stimulus that moves in their preferred direction, but respond weakly or do not respond to a stimulus that moves in the opposite or null direction. DS RGCs are sensitive to acetylcholine, and starburst amacrine cells (SACs) make cholinergic synapses on DS RGCs. We studied the distributions of nicotinic acetylcholine receptor (nAChR) α7 and β2 subunits on the dendritic arbors of DS RGCs to search for anisotropies that contribute to the directional preferences of DS RGCs. The DS RGCs from the retinas of postnatal mice (postnatal day P5, P10, and P15) were injected with Lucifer yellow, and injected cells were identified by their dendritic morphology. The dendrites of the DS RGCs were labeled with antibodies for either the nAChR α7 or β2 subunit as well as postsynaptic density protein-95 (PSD-95), visualized by confocal microscopy, and reconstructed from high-resolution confocal images. The distribution of nAChR subunits on the dendritic arbors in both the ON and OFF layers of the RGCs revealed an asymmetrical pattern on early postnatal day P5. However, the distributions of nAChR subunits on the dendritic arbors were not asymmetric on P10 and P15. Our results therefore provide anatomical and developmental evidence suggesting that the nAChR α7 and β2 subunits may involve in the early direction-selectivity formation of DS RGCs in the mouse retina.

Heteromeric Kv7.2/7.3 channels differentially regulate action potential initiation and conduction in neocortical myelinated axons.

Rapid energy-efficient signaling along vertebrate axons is achieved through intricate subcellular arrangements of voltage-gated ion channels and myelination. One recently appreciated example is the tight colocalization of K(v)7 potassium channels and voltage-gated sodium (Na(v)) channels in the axonal initial segment and nodes of Ranvier. The local biophysical properties of these K(v)7 channels and the functional impact of colocalization with Na(v) channels remain poorly understood. Here, we quantitatively examined K(v)7 channels in myelinated axons of rat neocortical pyramidal neurons using high-resolution confocal imaging and patch-clamp recording. K(v)7.2 and 7.3 immunoreactivity steeply increased within the distal two-thirds of the axon initial segment and was mirrored by the conductance density estimates, which increased from ~12 (proximal) to 150 pS μm(-2) (distal). The axonal initial segment and nodal M-currents were similar in voltage dependence and kinetics, carried by K(v)7.2/7.3 heterotetramers, 4% activated at the resting membrane potential and rapidly activated with single-exponential time constants (~15 ms at 28 mV). Experiments and computational modeling showed that while somatodendritic K(v)7 channels are strongly activated by the backpropagating action potential to attenuate the afterdepolarization and repetitive firing, axonal K(v)7 channels are minimally recruited by the forward-propagating action potential. Instead, in nodal domains K(v)7.2/7.3 channels were found to increase Na(v) channel availability and action potential amplitude by stabilizing the resting membrane potential. Thus, K(v)7 clustering near axonal Na(v) channels serves specific and context-dependent roles, both restraining initiation and enhancing conduction of the action potential.

The Relationship between Membrane Potential and Calcium Dynamics in Glucose-Stimulated Beta Cell Syncytium in Acute Mouse Pancreas Tissue Slices

Oscillatory electrical activity is regarded as a hallmark of the pancreatic beta cell glucose-dependent excitability pattern. Electrophysiologically recorded membrane potential oscillations in beta cells are associated with in-phase oscillatory cytosolic calcium activity ([Ca2+]i) measured with fluorescent probes. Recent high spatial and temporal resolution confocal imaging revealed that glucose stimulation of beta cells in intact islets within acute tissue slices produces a [Ca2+]i change with initial transient phase followed by a plateau phase with highly synchronized [Ca2+]i oscillations. Here, we aimed to correlate the plateau [Ca2+]i oscillations with the oscillations of membrane potential using patch-clamp and for the first time high resolution voltage-sensitive dye based confocal imaging. Our results demonstrated that the glucose-evoked membrane potential oscillations spread over the islet in a wave-like manner, their durations and wave velocities being comparable to the ones for [Ca2+]i oscillations and waves. High temporal resolution simultaneous records of membrane potential and [Ca2+]i confirmed tight but nevertheless limited coupling of the two processes, with membrane depolarization preceding the [Ca2+]i increase. The potassium channel blocker tetraethylammonium increased the velocity at which oscillations advanced over the islet by several-fold while, at the same time, emphasized differences in kinetics of the membrane potential and the [Ca2+]i. The combination of both imaging techniques provides a powerful tool that will help us attain deeper knowledge of the beta cell network.

Muscle histone deacetylase 4 upregulation in amyotrophic lateral sclerosis: potential role in reinnervation ability and disease progression

Amyotrophic lateral sclerosis is a typically rapidly progressive neurodegenerative disorder affecting motor neurons leading to progressive muscle paralysis and death, usually from respiratory failure, in 3-5 years. Some patients have slow disease progression and prolonged survival, but the underlying mechanisms remain poorly understood. Riluzole, the only approved treatment, only modestly prolongs survival and has no effect on muscle function. In the early phase of the disease, motor neuron loss is initially compensated for by collateral reinnervation, but over time this compensation fails, leading to progressive muscle wasting. The crucial role of muscle histone deacetylase 4 and its regulator microRNA-206 in compensatory reinnervation and disease progression was recently suggested in a mouse model of amyotrophic lateral sclerosis (transgenic mice carrying human mutations in the superoxide dismutase gene). Here, we sought to investigate whether the microRNA-206-histone deacetylase 4 pathway plays a role in muscle compensatory reinnervation in patients with amyotrophic lateral sclerosis and thus contributes to disease outcome differences. We studied muscle reinnervation using high-resolution confocal imaging of neuromuscular junctions in muscle samples obtained from 11 patients with amyotrophic lateral sclerosis, including five long-term survivors. We showed that the proportion of reinnervated neuromuscular junctions was significantly higher in long-term survivors than in patients with rapidly progressive disease. We analysed the expression of muscle candidate genes involved in the reinnervation process and showed that histone deacetylase 4 upregulation was significantly greater in patients with rapidly progressive disease and was negatively correlated with the extent of muscle reinnervation and functional outcome. Conversely, the proposed regulator of histone deacetylase 4, microRNA-206, was upregulated in both patient groups, but did not correlate with disease progression or reinnervation. We conclude that muscle expression of histone deacetylase 4 may be a key factor for muscle reinnervation and disease progression in patients with amyotrophic lateral sclerosis. Specific histone deacetylase 4 inhibitors may then constitute a therapeutic approach to enhancing motor performance and slowing disease progression in amyotrophic lateral sclerosis.

Dynamics of Akt activation during mouse embryo development: Distinct subcellular patterns distinguish proliferating versus differentiating cells

Akt is a highly conserved serine–threonine protein kinase which has been implicated in a wide variety of cellular functions, from the regulation of growth and metabolism, to activation of pro-survival pathways and cell proliferation, and promotion of differentiation in specific cell types. However, very little is known about the spatial and temporal pattern of Akt activity within cells and whether this pattern changes as cells enter and proceed in their differentiation programs. To address this issue we profiled Akt activation in E8.5–E13.5 mouse embryos and in C2C12 cells. We used a commercial antibody against Akt, phosphorylated on one of its activating residues, Thr-308, and performed high resolution confocal imaging of the immunofluorescence in labeled embryos. We observe strong Akt activity during mitosis in the dermomyotome, the neuroepithelium and some mesenchymal cells. This burst of activity fills the whole cell except for heterochromatin-positive areas in the nucleus. A surge in activity during mitosis is also observed in subconfluent C2C12 cells. Later on in the differentiation programs of skeletal muscle and neural cells, derivatives of the dermomyotome and neuroepithelium, respectively, we find robust, sustained Akt activity in the cytoplasm, but not in the nucleus. Concomitantly with skeletal muscle differentiation, Akt activity becomes concentrated in the sarcomeric Z-disks whereas developing neurons maintain a uniform cytoplasmic pattern of activated Akt. Our findings reveal unprecedented cellular and subcellular details of Akt activity during mouse embryo development, which is spatially and temporally consistent with proposed functions for Akt in mitosis and myogenic and neural differentiation and/or survival. Our results thus demonstrate a subcellular change in the pattern of Akt activation when skeletal muscle and neural progenitor cells cease dividing and progress in their differentiation programs.

Multimodal Assessment of Microscopic Morphology and Retinal Function in Patients With Geographic Atrophy

To correlate retinal function and visual sensitivity with retinal morphology revealed by ultrahigh-resolution imaging with adaptive optics-optical coherence tomography (AO-OCT), on patients with geographic atrophy. Five eyes from five subjects were tested (four with geographic atrophy [66.3 ± 6.4 years, mean ± 1 SD] and one normal [61 years]). Photopic and scotopic multifocal electroretinograms (mfERGs) were recorded. Visual fields were assessed with microperimetry (mP) combined with a scanning laser ophthalmoscope for high-resolution confocal retinal fundus imaging. The eye tracker of the microperimeter identified the preferred retinal locus that was then used as a reference for precise targeting of areas for advanced retinal imaging. Images were obtained with purpose-built, in-house, ultrahigh resolution AO-OCT. Fundus autofluorescence (FAF) and color fundus (CF) photographs were also acquired. The AO-OCT imaging provided detailed cross-sectional structural representation of the retina. Up to 12 retinal layers were identified in the normal subject while many severe retinal abnormalities (i.e., calcified drusen, drusenoid pigment epithelium detachment, outer retinal tubulation) were identified in the retinae of the GA patients. The functional tests showed preservation of sensitivities, although somewhat compromised, at the border of the GA. The images provided here advance our knowledge of the morphology of retinal layers in GA patients. While there was a strong correlation between altered retinal structure and reduction in visual function, there were a number of examples in which the photoreceptor inner/outer segment (IS/OS) junctions lost reflectivity at the margins of GA, while visual function was still demonstrated. This was shown to be due to changes in photoreceptor orientation near the GA border.

Angiophagy

Cell viability in the brain is critically dependent on uninterrupted blood flow through the microvasculature. However, microvessels, because of their small diameter and low-flow velocity, are prone to occlusion by blood clots, fragments of atheromatous plaques, or other circulating debris. Furthermore, microvascular occlusions can occur after large-vessel thrombosis because of blood stasis or due to clot fragmentation and distal microembolization. Microvascular occlusion could thus be a frequent phenomenon throughout life that may play important roles in the pathogenesis of cerebral ischemia and reperfusion, age-related vascular cognitive impairment, and cognitive decline after surgical procedures, such as coronary artery bypass graft. Given the propensity of microvessels to occlusion, it is likely that robust mechanisms have evolved to ensure microvascular patency. The main mechanism that prevents microvascular occlusion is constituted by the combined effects of the fibrinolytic system and hemodynamic pressure, which lead to embolus dissolution and washout. However, the fibrinolytic system is limited to breaking down fibrin-based blood clots and is unable to disrupt other materials, such as calcium and cholesterol crystals and cell debris, which are present in complex thrombi and may occlude microvessels throughout life. Using an experimental mouse model of internal carotid embolization with fluorescently labeled microemboli (10–60 µm), we were able to study the outcome of individual microvascular occlusions in the mouse brain. We tracked individual microvessels using high-resolution confocal imaging in histological brain preparations and by time-lapse 2 photon microscopy of the meninges and superficial cortex in living mice. Using these methods, we recently found a novel mechanism of microvascular recanalization, which we termed “angiophagy.” This mechanism involves the engulfment of emboli by the endothelium followed by their translocation through the vessel wall into the perivascular space. Angiophagy is a robust mechanism that leads to blood flow reestablishment within hours of the embolus extrusion.1 Embolus extravasation …

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP and Plp-GFP mice, respectively. Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact. Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species. In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation has proven useful for studies of NMJ development, physiology and pathology. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.