Optical Slices Research Articles

An analysis of the role of a retroendocytosis pathway in ABCA1-mediated cholesterol efflux from macrophages

The ATP binding cassette transporter A-1 (ABCA1) is critical for apolipoprotein-mediated cholesterol efflux, an important mechanism employed by macrophages to avoid becoming lipid-laden foam cells, the hallmark of early atherosclerotic lesions. It has been proposed that lipid-free apolipoprotein A-I (apoA-I) enters the cell and is resecreted as a lipidated particle via a retroendocytosis pathway during ABCA1-mediated cholesterol efflux from macrophages. To determine the functional importance of such a pathway, confocal microscopy was used to characterize the internalization of a fully functional apoA-I cysteine mutant containing a thiol-reactive fluorescent probe in cultured macrophages. ApoA-I was also endogenously labeled with (35)S-methionine to quantify cellular uptake and to determine the metabolic fate of the internalized protein. It was found that apoA-I was specifically taken inside macrophages and that a small amount of intact apoA-I was resecreted from the cells. However, a majority of the label that reappeared in the media was degraded. We estimate that the mass of apoA-I retroendocytosed is not sufficient to account for the HDL produced by the cholesterol efflux reaction. Furthermore, we have demonstrated that lipid-free apoA-I-mediated cholesterol efflux from macrophages can be pharmacologically uncoupled from apoA-I internalization into cells. On the basis these findings, we present a model in which the ABCA1-mediated lipid transfer process occurs primarily at the membrane surface in macrophages, but still accounts for the observed specific internalization of apoA-I.

Quantitative proliferation dynamics and random chromosome segregation of hair follicle stem cells

Regulation of stem cell (SC) proliferation is central to tissue homoeostasis, injury repair, and cancer development. Accumulation of replication errors in SCs is limited by either infrequent division and/or by chromosome sorting to retain preferentially the oldest 'immortal' DNA strand. The frequency of SC divisions and the chromosome-sorting phenomenon are difficult to examine accurately with existing methods. To address this question, we developed a strategy to count divisions of hair follicle (HF) SCs over time, and provide the first quantitative proliferation history of a tissue SC during its normal homoeostasis. We uncovered an unexpectedly high cellular turnover in the SC compartment in one round of activation. Our study provides quantitative data in support of the long-standing infrequent SC division model, and shows that HF SCs do not retain the older DNA strands or sort their chromosome. This new ability to count divisions in vivo has relevance for obtaining basic knowledge of tissue kinetics.

Reprint of “Stereology meets electron tomography: towards quantitative 3D electron microscopy” [J. Struct. Biol. 159 (2007) 443–450

Stereological tools are the gold standard for accurate (i.e., unbiased) and precise quantification of any microscopic sample. The past decades have provided a broad spectrum of tools to estimate a variety of parameters such as volumes, surfaces, lengths, and numbers. Some of them require pairs of parallel sections that can be produced by either physical or optical sectioning, with optical sectioning being much more efficient when applicable. Unfortunately, transmission electron microscopy could not fully profit from these riches, mainly because of the large depth of field. Hence, optical sectioning was a long-time desire for electron microscopists. This desire was fulfilled with the development of electron tomography that yield stacks of slices from electron microscopic sections. Now, parallel optical slices of a previously unimagined small thickness (2-5nm axial resolution) can be produced. These optical slices minimize problems related to overprojection effects, and allow for direct stereological analysis, e.g., volume estimation with the Cavalieri principle and number estimation with the optical disector method. Here, we demonstrate that the symbiosis of stereology and electron tomography is an easy and efficient way for quantitative analysis at the electron microscopic level. We call this approach quantitative 3D electron microscopy.

Changes produced by external radiation in parameters influencing intestinal permeability and microparticle uptake in vitro

Purpose: To determine the interaction between X-irradiation and in vitro intestinal microparticle uptake through Caco-2 epithelial cells.Methods: Caco-2 cells were cultured on 3 μm porous membranes for 21 days, X-irradiated with 2 Gy or sham-irradiated, then incubated for 5 or 30 min and exposed apically for 30 min to 2 μm latex microparticles. Measurements included cell dimensions, from confocal microscope ‘optical slices’; transepithelial resistance (TER) for tight junction (TJ) permeability; particle aggregation; and particle numbers on (adsorbed), in (intraepithelial) and through (submembranous) the epithelium.Results: Irradiation alone reduced TJ permeability more than sham-treatment, more so 5 min than 30 min after treatment. Irradiated epithelia were more permeable to particles than the equivalent sham-irradiated or previously untreated (particle only) groups: the latter two were similar. Irradiation altered adsorbed particle numbers and increased submembranous counts: particle uptake correlated best with cell height.Conclusions: 2 Gy X-irradiation increased particle uptake and translocation through the epithelium. This correlated well with the TJ opening seen after particle exposure in irradiated samples and changes in cell morphology. New data on cell dimensions underlined the similarity in particle uptake between this in vitro epithelium and that in an in vivo model, highlighting the translational significance of the work.

The Lipid Droplet Binding Domain of Hepatitis C Virus Core Protein Is a Major Determinant for Efficient Virus Assembly

Hepatitis C virus core protein forms the viral capsid and is targeted to lipid droplets (LDs) by its domain 2 (D2). By using a comparative analysis of two hepatitis C virus genomes (JFH1 and Jc1) differing in their level of virus production in cultured human hepatoma cells, we demonstrate that the core of the genotype 2a isolate J6 that is present in Jc1 mediates efficient assembly and release of infectious virions. Mapping studies identified a single amino acid residue in D2 as a major determinant for enhanced assembly and release of infectious Jc1 particles. Confocal microscopy analyses demonstrate that core protein in JFH1-replicating cells co-localizes perfectly with LDs and induces their accumulation in the perinuclear area, whereas no such accumulation of LDs and only a partial co-localization of core and LDs were found with the Jc1 genome. By using a fluorescence recovery after photobleaching assay, we found that green fluorescent protein-tagged D2 variants are mobile on LDs and that J6- and JFH1-D2 differ in their mobility. Taken together, our results demonstrate that the binding strength of the D2 domain of core for LDs is crucial for determining the efficiency of virus assembly.

Biocompatibility of Contact Lens Solutions Using Confocal Laser Scanning Microscopy and the In Vitro Bovine Cornea

This study used high-resolution research confocal laser scanning microscopy to study the epithelium of fresh bovine corneas treated with two contact lens solutions. Fresh bovine corneal buttons were stained with a combination of the cell-permeable mitochondrial- and nuclear-specific dyes rhodamine 123 and Hoechst 33342 or acridine orange and imaged with a confocal laser scanning microscope. High-resolution X,Y images of the corneal epithelium were taken with a Zeiss 510 confocal laser scanning microscope at a 0.44-062 -microm Z step, and three-dimensional images of the entire thickness of the corneal epithelium were reconstructed. Semiautomated quantitative MatLab software analysis was applied to count the mitochondria in each optical slice. The effects of treatment for 30 minutes with OPTI-FREE Express and ReNu MultiPlus No Rub were investigated and compared to Hanks' balanced salt solution controls. This study showed the feasibility of using the confocal laser scanning microscope with fluorescent dyes to study organelles throughout the epithelial layers of fresh unfixed bovine corneas. The mitochondria in the intermediate and basal epithelium are abundant and range in length from less than 1 microm to threads greater than 15 microm, with a tendency to accumulate around the nuclei. Image analysis indicated significantly fewer mitochondria only in the superficial epithelium after treatment with ReNu MultiPlus No Rub treatment. OPTI-FREE Express treatment produced significantly fewer mitochondria not only in the superficial but also in the intermediate epithelial layers. OPTI-FREE Express has the potential for deeper mitochondrial effects compared to ReNu MultiPlus No Rub and Hanks' balanced salt solution controls.

In vivo confocal laser laparoscopy allows real time subsurface microscopy in animal models of liver disease

Histopathology is essential in the diagnostic workup of most liver diseases. However, biopsy sampling might carry risks, is subject to sampling error, and does not provide dynamic tissue imaging. Therefore a newly developed miniaturised confocal probe was evaluated for in vivo microscopic imaging in rodent models of human liver diseases. The handheld laparoscopy probe used a 488nm single line laser for fluorophore excitation. Optical slice thickness was 7 microm, lateral resolution 0.7 microm. Imaging depth was 0-250 microm below the tissue surface. Imaging using different fluorescent staining protocols was performed in healthy mice, IFN-gamma- and IL-12-induced hepatitis, after bile duct ligation, and in ob/ob mice. Confocal imaging permitted microscopic imaging of the liver in vivo at high resolution. Landmarks of liver diseases such as inflammatory infiltration, steatosis, bile duct proliferation, pericellular fibrosis and perfusion anomalies could be characterised, localised and differentiated in vivo. In vivo confocal microscopy with this newly developed probe allows real-time subsurface imaging and characterisation of normal and pathologic liver tissue at high resolution. Since this technology is suited for laparoscopy in humans, it has the potential to impact on the diagnostic workup of liver diseases and to permit dynamic monitoring of (patho-)physiologic events in vivo.

Identification of Epithelial Gaps in Human Small and Large Intestine by Confocal Endomicroscopy

Confocal endomicroscopy is an emerging technology that poses the endoscopist with challenges for identifying epithelial structures in the human intestine. We have shown previously that the murine intestinal epithelium is punctuated by gaps caused by cell shedding. The goals of this study were to determine if confocal endomicroscopy could resolve the presence of human epithelial gaps and whether a proinflammatory cytokine could increase cell shedding. Intestinal mucosa was imaged after staining with acriflavine. Confocal endomicroscopy of 17 patients yielded 6277 images from the human terminal ileum and rectum. Results were validated by parallel studies of anesthetized mice (wild-type and Math1(DeltaIntestine)) using rigid confocal probe microscopy, 2-photon/confocal microscopy, and scanning electron microscopy. Human terminal ileal and rectal epithelium revealed unstained areas with the diameter of an individual epithelial cell, with 2 distinct morphologies. One had a "target" appearance, shown by mouse studies to be goblet cells. The other morphology had no nucleus and was observed by rigid confocal probe microscopy and scanning electron microscopy in the villi of Math1(DeltaIntestine) mice, which lack goblet cells. In the mouse, tumor necrosis factor alpha (0.33 microg/g intraperitoneally) increases cell shedding by 27-fold and caused loss of barrier function across 20% of resultant gaps. Confocal endomicroscopy can distinguish between epithelial discontinuities (gaps) and goblet cells in human intestine. Results suggest that the sealing of epithelial gaps must be considered as a component of the intestinal barrier and has potential implications for intestinal barrier dysfunction in human disease.

Stereology meets electron tomography: Towards quantitative 3D electron microscopy

Stereological tools are the gold standard for accurate (i.e., unbiased) and precise quantification of any microscopic sample. The past decades have provided a broad spectrum of tools to estimate a variety of parameters such as volumes, surfaces, lengths, and numbers. Some of them require pairs of parallel sections that can be produced by either physical or optical sectioning, with optical sectioning being much more efficient when applicable. Unfortunately, transmission electron microscopy could not fully profit from these riches, mainly because of the large depth of field. Hence, optical sectioning was a long-time desire for electron microscopists. This desire was fulfilled with the development of electron tomography that yield stacks of slices from electron microscopic sections. Now, parallel optical slices of a previously unimagined small thickness (2–5 nm axial resolution) can be produced. These optical slices minimize problems related to overprojection effects, and allow for direct stereological analysis, e.g., volume estimation with the Cavalieri principle and number estimation with the optical disector method. Here, we demonstrate that the symbiosis of stereology and electron tomography is an easy and efficient way for quantitative analysis at the electron microscopic level. We call this approach quantitative 3D electron microscopy.

In-vivo confocal real-time mini-microscopy in animal models of human inflammatory and neoplastic diseases

Although various improvements in tissue imaging modalities have recently been achieved, in-vivo molecular and subsurface imaging in the field of gastroenterology remains a technical challenge. In this study we evaluated a newly developed, handheld, miniaturized confocal laser microscopy probe for real-time in-vivo molecular and subsurface imaging in rodent models of human disease. The minimicroscope uses a 488-nm, single line laser for fluorophore excitation. The optical slice thickness is 7 microm, the lateral resolution 0.7 microm. The range of the z-axis is 0-250 microm below the tissue surface. Imaging was performed using different fluorescent staining protocols; 5-carboxyfluorescein-labeled octreotate was synthesized for targeted molecular imaging. Cellular and subcellular details of the gastrointestinal tract could be visualized in vivo at high resolution. Confocal real-time microscopy allowed in-vivo identification of tumor vessels and liver metastases, as well as diagnosis of focal hepatic inflammation, necrosis, and associated perfusion anomalies. Somatostatin-receptor targeting permitted in-vivo molecular staining of AR42-J-induced carcinoma and pancreatic islet cells. Confocal mini-microscopy allows rapid in-vivo molecular and subsurface imaging of normal and pathological tissue in the gastrointestinal tract at high resolution. Because this technology is applicable to humans, it might impact on future in-vivo microsocpic and molecular diagnosis of diseases such as cancer and inflammation.

The Inner Loop of Tetraspanins CD82 and CD81 Mediates Interactions with Human T Cell Lymphotrophic Virus Type 1 Gag Protein

The tetraspanin superfamily proteins play important roles in organizing membrane protein complexes, modulating integrin function, and controlling T cell adhesion. Tetraspanins such as CD82 contain two extracellular loops with its N terminus, C terminus, and inner loop exposed to the cytoplasm. The matrix (MA) domain of human T cell lymphotrophic virus, type 1 (HTLV-1), Gag interacts with the cytoplasmic face of the plasma membrane and is concentrated at tetraspanin-enriched microdomains. To understand the basis of this association, we generated site-directed mutations in the various domains of CD82 and used coimmunoprecipitation and colocalization approaches to examine interactions with HTLV-1 MA. The large extracellular loop of CD82, which is important for interactions with integrins, was not required for the association with HTLV-1 MA. The cytoplasmic N terminus and C terminus of CD82 were also dispensable for CD82-MA interactions. In contrast, mutations of conserved amino acids in the inner loop of CD82 or of palmitoylated cysteines that flank the inner loop diminished CD82 association with MA. HTLV-1 MA also interacted with the inner loop of CD81. Thus, association of HTLV-1 Gag with tetraspanin-enriched microdomains is mediated by the inner loops of CD81 and CD82.

In vivosubsurface morphological and functional cellular and subcellular imaging of the gastrointestinal tract with confocal mini-microscopy

To evaluate a newly developed hand-held confocal probe for in vivo microscopic imaging of the complete gastrointestinal tract in rodents. A novel rigid confocal probe (diameter 7 mm) was designed with optical features similar to the flexible endomicroscopy system for use in humans using a 488 nm single line laser for fluorophore excitation. Light emission was detected at 505 to 750 nm. The field of view was 475 microm multiply 475 microm. Optical slice thickness was 7 microm with a lateral resolution of 0.7 microm. Subsurface serial images at different depths (surface to 250 microm) were generated in real time at 1024 multiply 1024 pixels (0.8 frames/s) by placing the probe onto the tissue in gentle, stable contact. Tissue specimens were sampled for histopathological correlation. The esophagus, stomach, small and large intestine and meso, liver, pancreas and gall bladder were visualised in vivo at high resolution in n = 48 mice. Real time microscopic imaging with the confocal mini-microscopy probe was easy to achieve. The different staining protocols (fluorescein, acriflavine, FITC-labelled dextran and L. esculentum lectin) each highlighted specific aspects of the tissue, and in vivo imaging correlated excellently with conventional histology. In vivo blood flow monitoring added a functional quality to morphologic imaging. Confocal microscopy is feasible in vivo allowing the visualisation of the complete GI tract at high resolution even of subsurface tissue structures. The new confocal probe design evaluated in this study is compatible with laparoscopy and significantly expands the field of possible applications to intra-abdominal organs. It allows immediate testing of new in vivo staining and application options and therefore permits rapid transfer from animal studies to clinical use in patients.

Arrestin Is Required for Agonist-induced Trafficking of Voltage-dependent Calcium Channels

Many metabotropic receptors in the nervous system act through signaling pathways that result in the inhibition of voltage-dependent calcium channels. Our previous findings showed that activation of seven-transmembrane receptors results in the internalization of calcium channels. This internalization takes place within a few seconds, raising the question of whether the endocytic machinery is in close proximity to the calcium channel to cause such rapid internalization. Here we show that voltage-dependent calcium channels are pre-associated with arrestin, a protein known to play a role in receptor trafficking. Upon GABAB receptor activation, receptors are recruited to the arrestin-channel complex and internalized. beta-Arrestin 1 selectively binds to the SNARE-binding region of the calcium channel. Peptides containing the arrestin-binding site of the channel disrupt agonist-induced channel internalization. Taken together these data suggest a novel neuronal role for arrestin.

Preparation and characterisation of fluorescent polystyrene spheres coated with different sized quantum dots

Water soluble CdSe quantum dots (QDs) coated polymer sphere with single and multiple fluorescent peaks have been prepared through the layer by layer self-assembly method. Polystyrene (PS) spheres ranging mainly from 330 to 750 nm and from 1·0 to 3·5 μm were used as the templates for deposition of three sized fluorescent QDs. The results of transmission electron microscopy and fluorescent confocal optical slice showed that the PS/CdSe composites were spherical in shape with core–shell structures. Emission colours of the mixture of spheres with single colour signal emitting at different wavelengths stimulated by a single light source could be distinguished clearly. Single spheres with separated, multiple fluorescence peaks could be easily obtained by controlling the ratios of the three sized CdSe QDs.

Different Domains of the AMPA Receptor Direct Stargazin-mediated Trafficking and Stargazin-mediated Modulation of Kinetics

Stargazin is an accessory protein of AMPA receptors that enhances surface expression and also affects the biophysical properties of the receptor. AMPA receptor domains necessary for either of these two processes have not yet been identified. Here, we used confocal imaging and electrophysiology of heterologously expressed, fluorophore-tagged GluR1, GluR2, and stargazin to study surface expression and desensitization kinetics. Stargazin-mediated trafficking was sensitive to the nature of the AMPA receptor cytoplasmic domain. The insertion of YFP after residue 15 of the truncated cytoplasmic tail of GluR1i perturbed stargazin-mediated trafficking of the receptor but not its modulation of desensitization kinetics. This construct also failed to permit fluorescence resonance energy transfer (FRET) with stargazin in the endoplasmic reticulum (ER), whereas FRET between fluorophore-tagged stargazin and non-truncated AMPA receptors demonstrated a specific interaction between these proteins, both in the ER and the plasma membrane. Rather than encoding a specific binding site, the fluorophore-tagged C terminus may restrict access to one or more ER retention sites. Although perturbations of the C terminus impeded stargazin-mediated trafficking to the plasma membrane, the effects of stargazin on the biophysical properties of AMPA receptors (i.e. modulation of desensitization) remained intact. These data provide strong evidence that the AMPA receptor domains required for stargazin modulation of gating and trafficking are separable.

Optimising non‐viral gene delivery in a tumour spheroid model

Our current understanding of how the unique tumour microenvironment influences the efficacy of gene delivery is limited. The current investigation systematically examines the efficiency of several non-viral gene transfer agents to transfect multicellular tumour spheroids (MCTS), an in vitro model that displays a faithful three-dimensional (3D) representation of solid tumour tissue. Using a luciferase reporter assay, gene transfer to MCTS was optimised for 22 kDa linear and 25 kDa branched polyethyleneimine (PEI), the cationic lipids Lipofectamine(trade mark) and DCChol : DOPE, and the physical approach of tissue electroporation. Confocal microscopy was used to take optical tissue slices to identify the tissue localisation of green fluorescent protein (GFP) reporter gene expression and the distribution of fluorescently labelled complexes. A MCTS model of quiescent tumour regions was used to establish the influence of cellular proliferation status on gene transfer efficiency. Of the polyplexes tested, 22 kDa linear PEI provided optimal gene delivery, with gene expression peaking at 46 h. Despite being the optimal vector tested, PEI-mediated transfection was limited to cells at the MCTS periphery. Using fluorescent PEI, it was found that complexes could only penetrate the outer 3-5 proliferating cell layers of the MCTS, sparing the deeper quiescent cells. Gene delivery in an MCTS model comprised entirely of quiescent cells demonstrated that in addition to being inaccessible to the vector, quiescent tumour regions are inherently less susceptible to PEI-mediated transfection than proliferating regions. This 'resistance' to transfection observed in quiescent cells was overcome through the use of electroporation. Despite the improved efficacy of electroporation in quiescent tissue, the gene expression was still confined to the outer regions of MCTS. The results suggest that limited access to central regions of an MCTS remain a significant barrier to gene delivery. This data provides new insights into tumour-specific factors affecting non-viral gene transfer and highlights the difficulties in delivering genes to avascular tumour regions. The MCTS model is a useful system for the initial screening of future gene therapy strategies for solid tumours.

Imaging of Dynamic Secretory Vesicles in Living Pollen Tubes of Picea meyeri Using Evanescent Wave Microscopy

Evanescent wave excitation was used to visualize individual, FM4-64-labeled secretory vesicles in an optical slice proximal to the plasma membrane of Picea meyeri pollen tubes. A standard upright microscope was modified to accommodate the optics used to direct a laser beam at a variable angle. Under evanescent wave microscopy or total internal reflection fluorescence microscopy, fluorophores localized near the surface were excited with evanescent waves, which decay exponentially with distance from the interface. Evanescent waves with penetration depths of 60 to 400 nm were generated by varying the angle of incidence of the laser beam. Kinetic analysis of vesicle trafficking was made through an approximately 300-nm optical section beneath the plasma membrane using time-lapse evanescent wave imaging of individual fluorescently labeled vesicles. Two-dimensional trajectories of individual vesicles were obtained from the resulting time-resolved image stacks and were used to characterize the vesicles in terms of their average fluorescence and mobility, expressed here as the two-dimensional diffusion coefficient D2. The velocity and direction of vesicle motions, frame-to-frame displacement, and vesicle trajectories were also calculated. Analysis of individual vesicles revealed for the first time, to our knowledge, that two types of motion are present, and that vesicles in living pollen tubes exhibit complicated behaviors and oscillations that differ from the simple Brownian motion reported in previous investigations. Furthermore, disruption of the actin cytoskeleton had a much more pronounced effect on vesicle mobility than did disruption of the microtubules, suggesting that actin cytoskeleton plays a primary role in vesicle mobility.

Simultaneous Monitoring of Ionophore- and Inhibitor-mediated Plasma and Mitochondrial Membrane Potential Changes in Cultured Neurons

Although natural and synthetic ionophores are widely exploited in cell studies, for example, to influence cytoplasmic free calcium concentrations and to depolarize in situ mitochondria, their inherent lack of membrane selectivity means that they affect the ion permeability of both plasma and mitochondrial membranes. A similar ambiguity affects the interpretation of signals from fluorescent membrane-permeant cations (usually termed "mitochondrial membrane potential indicators"), because the accumulation of these probes is influenced by both plasma and mitochondrial membrane potentials. To resolve some of these problems a technique is developed to allow simultaneous monitoring of plasma and mitochondrial membrane potentials at single-cell resolution using a cationic and anionic fluorescent probe. A computer program is described that transforms the fluorescence changes into dynamic estimates of changes in plasma and mitochondrial potentials. Exploiting this technique, primary cultures of rat cerebellar granule neurons display a concentration-dependent response to ionomycin: low concentrations mimic nigericin by hyperpolarizing the mitochondria while slowly depolarizing the plasma membrane and maintaining a stable elevated cytoplasmic calcium. Higher ionomycin concentrations induce a stochastic failure of calcium homeostasis that precedes both mitochondrial depolarization and an enhanced rate of plasma membrane depolarization. In addition, the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone only selectively depolarizes mitochondria at submicromolar concentrations. ATP synthase reversal following respiratory chain inhibition depolarizes the mitochondria by 26 mV.

Microscopic Changes in Non Erosive Reflux Disease (NERD) Can Be Diagnosed During Ongoing Endoscopy By Confocal Laser Endomicroscopy (CLE)

Introduction: Non erosive reflux disease (NERD) is the most frequent endoscopic finding in patients with gastroesophageal reflux disease (GERD). NERD is histological characterised by elongated papillae, basal cell proliferation and dilated intercellular spaces within the squamous cell epithelium in the distal esophagus. Aim of the current study was to investigate the value of confocal laser endomicroscopy for diagnosing microscopic changes in patients with NERD during ongoing upper endoscopy. Methods: Patients with reflux symptoms scheduled for upper endoscopy were examined with the newly developed confocal laser endoscope (Pentax, Tokyo, EC-3870CIFK). Fluorescein was applied intravenously to enable confocal endomicroscopy of the esophageal mucosa (0-250μm). Confocal images from macroscopic normal apparent mucosa were obtained from every quadrant at the Z-Line and 2 cm above. Confocal image data were collected at a scan rate of 1.6 sec/frames (1024 × 1024 pixels) using an optical slice thickness of 7μm (lateral resolution ≤ 1 μm). The degree of dilated intercellular spaces as well as the amount of capillary loops within the upper third of the mucosal layer was graduated during ongoing endoscopy. Results: 68 patients with GERD were included. 38 patients had to be excluded due to the presence of esophagitis or Barrett's epithelium. In 30 patients cellular and vascular changes within the distal part of the esophagus could be visualised in vivo at high resolution. The capillary loops within elongated papillae were readily visible due to the high contrast of fluorescein and the absence of vasculature within squamous cell epithelium. More than 5 capillary loops within the endomicroscopic field of view (500 × 500 μm) and dilated intercellular spaces (≥ 7 μm) predicted microscopic changes with a sensitivity of 94.9% and specificity of 85.4%, respectively (accuracy 91.7%). Conclusions: Confocal laser endomicroscopy can be used to identify microscopic changes in patients with NERD during ongoing upper endoscopy. Microscopic changes are often located at the 3 o'clock position 2cm above the Z-line. CLE enables targeted biopsies and may help to accurately diagnose patients with NERD and analyze the effect of PPI therapy on microscopic changes in patients with NERD in future studies.

Application of Phase Correlation to the Montage Synthesis and Three-Dimensional Reconstruction of Large Tissue Volumes from Confocal Laser Scanning Microscopy

We have implemented and tested a new automatic method for the montage synthesis and three-dimensional (3D) reconstruction of large tissue volumes from confocal laser scanning microscopy data (CLSM). This method relies on maximization of the phase correlation between adjacent images. It was tested on a large specimen (a murine heart) that was cut into a number of individual sections with thickness appropriate for CLSM. The sections were scanned horizontally (in-plane) and vertically (perpendicular to the optical planes) to produce "tiles" of a 3D volume. Phase correlation maximization was applied to the montage synthesis of in-plane tiles and 3D alignment of optical slices within a given physical section. The performance of the new method is evaluated.