Confocal Microscope System Research Articles

A Functional Role for N‐cadherin in Mediating Mechanotransduction in Arteriolar Vascular Smooth Muscle Cells

A hybrid atomic force (AFM)‐confocal microscopy system was used to examine whether mechanical forces applied through N‐cadherin adhesions on vascular smooth muscle cells (VSMC) could induce i) cellular contraction and ii) signaling events. AFM probes were modified by attaching a borosilicate microsphere (5 μm) to the tip and coating with either 1) a recombinant N‐cadherin chimera fusion protein (rhNcad), or 2) an N‐cadherin antibody; or 3) attaching a live VSMC to the end of the AFM cantilever. In all 3 cases, a strong adhesion was formed between the AFM probe and cell membrane; and AFM pulling forces (~1000 pN) induced a strong VSMC contractile response that pulled the probe oppositely. In addition, clustering of N‐cadherin‐GFP and RhoA‐GFP was observed when an rhNcad coated AFM probe was engaged on the VSMC membrane, and application of AFM pulling force enhanced the clustering. Immuno‐fluorescence labeling showed a dramatic increase of N‐cadherin clustering in the vessel wall of pressurized arterioles that developed spontaneous myogenic tone compared to unpressurized vessels, as well as spatial correlations between the N‐cadherin and the clusters of active RhoA, myosin II and the phosphorylated myosin phosphatase targeting subunit. Collectively, these results support an important role for N‐cadherin in mediating the myogenic mechanotransduction in VSMC. (AHA 0835676N to Z.S. and NIH P01HL095486 to G.A.M.)

Hypercapnia Induces Cleavage and Nuclear Localization of RelB Protein, Giving Insight into CO2 Sensing and Signaling

Carbon dioxide (CO(2)) is increasingly being appreciated as an intracellular signaling molecule that affects inflammatory and immune responses. Elevated arterial CO(2) (hypercapnia) is encountered in a range of clinical conditions, including chronic obstructive pulmonary disease, and as a consequence of therapeutic ventilation in acute respiratory distress syndrome. In patients suffering from this syndrome, therapeutic hypoventilation strategy designed to reduce mechanical damage to the lungs is accompanied by systemic hypercapnia and associated acidosis, which are associated with improved patient outcome. However, the molecular mechanisms underlying the beneficial effects of hypercapnia and the relative contribution of elevated CO(2) or associated acidosis to this response remain poorly understood. Recently, a role for the non-canonical NF-κB pathway has been postulated to be important in signaling the cellular transcriptional response to CO(2). In this study, we demonstrate that in cells exposed to elevated CO(2), the NF-κB family member RelB was cleaved to a lower molecular weight form and translocated to the nucleus in both mouse embryonic fibroblasts and human pulmonary epithelial cells (A549). Furthermore, elevated nuclear RelB was observed in vivo and correlated with hypercapnia-induced protection against LPS-induced lung injury. Hypercapnia-induced RelB processing was sensitive to proteasomal inhibition by MG-132 but was independent of the activity of glycogen synthase kinase 3β or MALT-1, both of which have been previously shown to mediate RelB processing. Taken together, these data demonstrate that RelB is a CO(2)-sensitive NF-κB family member that may contribute to the beneficial effects of hypercapnia in inflammatory diseases of the lung.

Synthesis and Characterization of Multifunctional Iron Oxide Nanoparticles

In order to get high water solubility, monodisperse, superparamagnetic nanoparticles, poly (acrylic acid) was employed to modify Fe3O4 by a high-temperature solution-phase hydrolysis approach. Then, folic acid (FA) and fluorescein isothiocyanate were successively conjugated with prepared magnetic nanoparticles (MNPs). The functional MNPs were characterized by X-ray diffraction (XRD), dynamic light scattering (DLS), transmission electron microscope (TEM), inductively coupled plasma-atomic emission spectrometer (ICP-AES), and vibrating sample magnetometer (VSM), respectively. The toxicity of the materials was evaluated by selecting NIH/3T3 fibroblast cells and no toxic effect was observed. The fluorescent imaging and targeting property of the MNPs were also realized in vitro and in vivo experiments by confocal laser scanning microscopy (CLSM) and Kodak In-Vivo FX Professional Imaging System, respectively. The results indicated that the final products exhibited interesting magnetic, optical and targeting properties for further potential applications in biological and biomedical fields.

Effect of astragalosideIV against rat myocardial cell apoptosis induced by oxidative stress via mitochondrial ATP-sensitive potassium channels.

Astragaloside is one of the most common traditional Chinese medicines and is derived from Astragalus membranaceus. Astragaloside IV (AsIV) is a monomer located in an extract of astragaloside. The current study investigated the protective effects of AsIV against hydrogen peroxide (H2O2)-induced injury in cardiocytes and elucidated the mechanisms responsible for this protective effect. Cultured neonatal rat cardiocytes were divided into five experimental groups as follows: i) Dimethyl sulfoxide; ii) H2O2; iii) AsIV+H2O2; iv) AsIV+H2O2+5-hydroxydecanoate (5-HD); and v) nicorandil+H2O2. Cardiocyte survival was analyzed using an MTT assay. Lactate dehydrogenase (LDH) release was also assessed to evaluate the viability of the cells. Intracellular reactive oxygen species (ROS) were measured by 2,7-dichlorodihydrofluorescein diacetate staining. The apoptotic rate was measured by flow cytometry. Mitochondrial membrane potential (ΔΨm) and intracellular calcium were observed using a laser confocal microscopy system. The results indicated that AsIV promoted the survival of cardiocytes (P<0.05), attenuated LDH release (P<0.05), ROS production (P<0.01) and apoptosis (P<0.01), stabilized the ΔΨm and reduced intracellular calcium overload (P<0.01) compared with the H2O2 group. The mitochondrial adenosine triphosphate-sensitive potassium channel (mitoKATP) inhibitor 5-HD was observed to partially reverse the protective effect of AsIV. Following treatment with 5-HD, the survival of cardiocytes was reduced (P<0.05), LDH release (P<0.01) and ROS production (P<0.05) were stimulated, ΔΨm and intracellular calcium change were increased (P<0.01) and apoptosis was increased (P<0.01) compared with the AsIV+H2O2 group. Thus, AsIV has potential for use in the suppression of apoptosis resulting from H2O2 exposure, and mitoKATP activation may underlie this protective mechanism.

Capillary density changes in rat femur from youth to aging

Background: The aging process, bone remodeling, and risks of fracture were associated with alteration of blood perfusion in the bone. However, capillary changes during aging in the bone have not been examined due to methodological difficulties. Objective: Investigate changes of capillary density and length during aging in rat femur using intravital confocal laser microscopy. Methods: Two-month male Wistar rats were divided into three groups: young (aged 4 to 6 months), adult (aged 12 to 14 months), and aged group (aged 20 to 22 months). The microvasculature of femur periosteum of each rat was visualized with FITC-labeled dextran, and observed via the window chamber under a confocal laser microscopic system. Capillary diameters, lengths, and densities were measured based on the obtained images. Capillary changes (density and length) during aging were analyzed. Results: The mean capillary density increases up to 130% from young to adult, but decreases up to 15% from adult to aged. Capillary densities against diameter were in a near-normal distribution with a maximum of 130, 250, and 15/mm2 at 3 to 5, 3 to 5, and 5 to 7 ?m diameter in the young, adult and aged group, respectively. The capillary length in the adult group was much shorter than those in the young and aged groups Conclusion: The capillary density in the rat femur changes from youth to aging. This can alter blood perfusion and result in bone changes. Keywords: Aging, capillary density, femur periosteum, fluorescence visualization, intravital observation, laser confocal microscopy

Capillary density changes in rat femur from youth to aging

Background: The aging process, bone remodeling, and risks of fracture were associated with alteration of blood perfusion in the bone. However, capillary changes during aging in the bone have not been examined due to methodological difficulties. Objective: Investigate changes of capillary density and length during aging in rat femur using intravital confocal laser microscopy. Methods: Two-month male Wistar rats were divided into three groups: young (aged 4 to 6 months), adult (aged 12 to 14 months), and aged group (aged 20 to 22 months). The microvasculature of femur periosteum of each rat was visualized with FITC-labeled dextran, and observed via the window chamber under a confocal laser microscopic system. Capillary diameters, lengths, and densities were measured based on the obtained images. Capillary changes (density and length) during aging were analyzed. Results: The mean capillary density increases up to 130% from young to adult, but decreases up to 15% from adult to aged. Capillary densities against diameter were in a near-normal distribution with a maximum of 130, 250, and 15/mm2 at 3 to 5, 3 to 5, and 5 to 7 ?m diameter in the young, adult and aged group, respectively. The capillary length in the adult group was much shorter than those in the young and aged groups Conclusion: The capillary density in the rat femur changes from youth to aging. This can alter blood perfusion and result in bone changes. Keywords: Aging, capillary density, femur periosteum, fluorescence visualization, intravital observation, laser confocal microscopy

Nuclear Envelope Phosphatase 1-Regulatory Subunit 1 (Formerly TMEM188) Is the Metazoan Spo7p Ortholog and Functions in the Lipin Activation Pathway

Lipin-1 catalyzes the formation of diacylglycerol from phosphatidic acid. Lipin-1 mutations cause lipodystrophy in mice and acute myopathy in humans. It is heavily phosphorylated, and the yeast ortholog Pah1p becomes membrane-associated and active upon dephosphorylation by the Nem1p-Spo7p membrane complex. A mammalian ortholog of Nem1p is the C-terminal domain nuclear envelope phosphatase 1 (CTDNEP1, formerly "dullard"), but its Spo7p-like partner is unknown, and the need for its existence is debated. Here, we identify the metazoan ortholog of Spo7p, TMEM188, renamed nuclear envelope phosphatase 1-regulatory subunit 1 (NEP1-R1). CTDNEP1 and NEP1-R1 together complement a nem1Δspo7Δ strain to block endoplasmic reticulum proliferation and restore triacylglycerol levels and lipid droplet number. The two human orthologs are in a complex in cells, and the amount of CTDNEP1 is increased in the presence of NEP1-R1. In the Caenorhabditis elegans embryo, expression of nematode CTDNEP1 and NEP1-R1, as well as lipin-1, is required for normal nuclear membrane breakdown after zygote formation. The expression pattern of NEP1-R1 and CTDNEP1 in human and mouse tissues closely mirrors that of lipin-1. CTDNEP1 can dephosphorylate lipins-1a, -1b, and -2 in human cells only in the presence of NEP1-R1. The nuclear fraction of lipin-1b is increased when CTDNEP1 and NEP1-R1 are co-expressed. Therefore, NEP1-R1 is functionally conserved from yeast to humans and functions in the lipin activation pathway.

HIV-1 Tat Protein Promotes Neuronal Dysfunction through Disruption of MicroRNAs

Over the last decade, small noncoding RNA molecules such as microRNAs (miRNAs) have emerged as critical regulators in the expression and function of eukaryotic genomes. It has been suggested that viral infections and neurological disease outcome may also be shaped by the influence of small RNAs. This has prompted us to suggest that HIV infection alters the endogenous miRNA expression patterns, thereby contributing to neuronal deregulation and AIDS dementia. Therefore, using primary cultures and neuronal cell lines, we examined the impact of a viral protein (HIV-1 Tat) on the expression of miRNAs due to its characteristic features such as release from the infected cells and taken up by noninfected cells. Using microRNA array assay, we demonstrated that Tat deregulates the levels of several miRNAs. Interestingly, miR-34a was among the most highly induced miRNAs in Tat-treated neurons. Tat also decreases the levels of miR-34a target genes such as CREB protein as shown by real time PCR. The effect of Tat was neutralized in the presence of anti-miR-34a. Using in situ hybridization assay, we found that the levels of miR-34a increase in Tat transgenic mice when compared with the parental mice. Therefore, we conclude that deregulation of neuronal functions by HIV-1 Tat protein is miRNA-dependent.

Theoretical and experimental studies on tightly focused vector vortex beams

The high-NA focusing properties of vector vortex beams are studied theoretically and experimentally. The vector vortex beams are generated by space-variant segmented subwavelength metallic gratings first. Then the mathematical expressions for the focused fields are derived based on the vector diffraction theory, and some numerical simulations are presented that show that the focused fields are not dark at the center and the focusing spot size of vector vortex beams with high topological charges approaches the diffraction limitation at high NA. Finally, to verify the theoretical analysis, the tightly focused fields are measured based on a confocal microscopy system when the NA of the objective lens is 0.90. The research results confirm the potential of vector vortex beams in some applications, such as optical trapping, laser printing, lithography, and material processing.

Infrasound increases intracellular calcium concentration and induces apoptosis in hippocampi of adult rats

In the present study, we determined the effect of infrasonic exposure on apoptosis and intracellular free Ca²⁺ ([Ca²⁺]i) levels in the hippocampus of adult rats. Adult rats were randomly divided into the control and infrasound exposure groups. For infrasound treatment, animals received infrasonic exposure at 90 (8 Hz) or 130 dB (8 Hz) for 2 h per day. Hippocampi were dissected, and isolated hippocampal neurons were cultured. The [Ca²⁺]i levels in hippocampal neurons from adult rat brains were determined by Fluo-3/AM staining with a confocal microscope system on days 1, 7, 14, 21 and 28 following infrasonic exposure. Apoptosis was evaluated by Annexin V-FITC and propidium iodide double staining. Positive cells were sorted and analyzed by flow cytometry. Elevated [Ca²⁺]i levels were observed on days 14 and 21 after rats received daily treatment with 90 or 130 dB sound pressure level (SPL) infrasonic exposure (p<0.01 vs. control). The highest levels of [Ca²⁺]i were detected in the 130 dB SPL infrasonic exposure group. Meanwhile, apoptosis in hippocampal neurons was found to increase on day 7 following 90 dB SPL infrasound exposure, and significantly increased on day 14. Upon 130 dB infrasound treatment, apoptosis was first observed on day 14, whereas the number of apoptotic cells gradually decreased thereafter. Additionally, a marked correlation between cell apoptosis and [Ca²⁺]i levels was found on day 14 and 21 following daily treatment with 90 and 130 dB SPL, respectively. These results demonstrate that a period of infrasonic exposure induced apoptosis and upregulated [Ca²⁺]i levels in hippocampal neurons, suggesting that infrasound may cause damage to the central nervous system (CNS) through the Ca²⁺‑mediated apoptotic pathway in hippocampal neurons.

Potentiation of Helicobacter pylori CagA Protein Virulence through Homodimerization

Chronic infection with Helicobacter pylori cagA-positive strains is associated with atrophic gastritis, peptic ulceration, and gastric carcinoma. The cagA gene product, CagA, is delivered into gastric epithelial cells via type IV secretion, where it undergoes tyrosine phosphorylation at the EPIYA motifs. Tyrosine-phosphorylated CagA binds and aberrantly activates the oncogenic tyrosine phosphatase SHP2, which mediates induction of elongated cell morphology (hummingbird phenotype) that reflects CagA virulence. CagA also binds and inhibits the polarity-regulating kinase partitioning-defective 1 (PAR1)/microtubule affinity-regulating kinase (MARK) via the CagA multimerization (CM) sequence independently of tyrosine phosphorylation. Because PAR1 exists as a homodimer, two CagA proteins appear to be passively dimerized through complex formation with a PAR1 dimer in cells. Interestingly, a CagA mutant that lacks the CM sequence displays a reduced SHP2 binding activity and exhibits an attenuated ability to induce the hummingbird phenotype, indicating that the CagA-PAR1 interaction also influences the morphological transformation. Here we investigated the role of CagA dimerization in induction of the hummingbird phenotype with the use of a chemical dimerizer, coumermycin. We found that CagA dimerization markedly stabilizes the CagA-SHP2 complex and thereby potentiates SHP2 deregulation, causing an increase in the number of hummingbird cells. Protrusions of hummingbird cells induced by chemical dimerization of CagA are further elongated by simultaneous inhibition of PAR1. This study revealed a role of the CM sequence in amplifying the magnitude of SHP2 deregulation by CagA, which, in conjunction with the CM sequence-mediated inhibition of PAR1, evokes morphological transformation that reflects in vivo CagA virulence.

Modeling Oculopharyngeal Muscular Dystrophy in Myotube Cultures Reveals Reduced Accumulation of Soluble Mutant PABPN1 Protein

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant disease caused by an alanine tract expansion mutation in poly(A) binding protein nuclear 1 (expPABPN1). To model OPMD in a myogenic and physiological context, we generated mouse myoblast cell clones stably expressing either human wild type (WT) or expPABPN1 at low levels. Transgene expression is induced on myotube differentiation and results in formation of insoluble nuclear PABPN1 aggregates that are similar to those observed in patients with OPMD. Quantitative analysis of PABPN1 in myotube cultures revealed that expPABPN1 accumulation and aggregation is greater than that of the WT protein. We found that aggregation of expPABPN1 is more affected than WT PABPN1 by inhibition of proteasome activity. Consistent with this, in myotube cultures expressing expPABPN1, deregulation of the proteasome was identified as the most significantly perturbed pathway. Differences in the accumulation of soluble WT and expPABPN1 were consistent with differences in ubiquitination and rate of protein turnover. This study demonstrates, for the first time to our knowledge, that, in myotubes, the ratio of soluble/insoluble expPABPN1 is significantly lower compared with that of the WT protein. We suggest that this difference can contribute to muscle weakness in OPMD.

Exciton-plasmon-photon conversion in silver nanowire: Polarization dependence

Polarization dependence of the exciton-plasmon-photon conversion in silver nanowire-quantum dots structure was investigated using a scanning confocal microscope system. We found that the fluorescence enhancement of the CdSe nanocrystals was correlated with the angle between the excitation light polarization and the silver nanowire direction. The polarization of the emission was also related with the nanowire direction. It was in majority in the direction parallel with nanowire due to the nano-antenna effect.

Motion compensated fiber-optic confocal microscope based on a common-path optical coherence tomography distance sensor

A motion compensated fiber-optic confocal microscope system is demonstrated by utilizing a Fourier domain common-path optical coherence tomography (FD-CP-OCT) distance sensor and a high-speed linear motor at the distal end of a fiber-optic confocal microscope imaging probe. The fiber-optic confocal microscope is based on a 460 μm diameter imaging bundle with 10 K cores. The fiber bundle was terminated with a gradient index lens system. Using one-dimensional A-scan data of CP-OCT, the distance deviation of the target from the focal plane of the probe was monitored and motion compensated at the rate of 840 Hz, for a confocal microscopic imaging frame rate of 1fps and was 200 × 200 pixels in size, with a distance error less than 5 μm.

Masked illumination scheme for a galvanometer scanning high‐speed confocal fluorescence microscope

High-speed beam scanning and data acquisition in a laser scanning confocal microscope system are normally implemented with a resonant galvanometer scanner and a frame grabber. However, the nonlinear scanning speed of a resonant galvanometer can generate nonuniform photobleaching in a fluorescence sample as well as image distortion near the edges of a galvanometer scanned fluorescence image. Besides, incompatibility of signal format between a frame grabber and a point detector can lead to digitization error during data acquisition. In this article, we introduce a masked illumination scheme which can effectively decrease drawbacks in fluorescence images taken by a laser scanning confocal microscope with a resonant galvanometer and a frame grabber. We have demonstrated that the difference of photobleaching between the center and the edge of a fluorescence image can be reduced from 26 to 5% in our confocal laser scanning microscope with a square illumination mask. Another advantage of our masked illumination scheme is that the zero level or the lowest input level of an analog signal in a frame grabber can be accurately set by the dark area of a mask in our masked illumination scheme. We have experimentally demonstrated the advantages of our masked illumination method in detail.

Experiments and quantitative analysis of frequency division multiplexing confocal fluorescence microscopy with UV excitation

In this paper, we experimentally demonstrated a two-channel frequency division multiplexing confocal fluorescence microscopy system using a UV laser as the excitation source. In our two-channel frequency division multiplexing confocal fluorescence system, the incoming laser beam was divided into two beams and each beam was modulated with an individual carrier frequency. These two laser beams were then spatially combined with a small angle and focused into two diffraction-limited spots on the targeted cell (rat neural cell) surface to generate fluorescent signal. As a result, the fluorescent signals from two spots of the rat neural cell surface can be demodulated and distinguished during data processing. Furthermore, a quantitative analysis on the cross-talk among different frequencies was provided as well. The experimental results confirm that the two-channel frequency division multiplexing confocal fluorescence technology can not only maintain the high spatial resolution, but also realize the multiple points detection simultaneously with high temporal resolution (within millisecond level range), which benefits the dynamic studies of living biological cells.

Aorta Fluorescence Imaging by Using Confocal Microscopy

The activated leukocyte attacked the vascular endothelium and the associated increase in VEcadherin number was observed in experiments. The confocal microscopic system with a prism-based wavelength filter was used for multiwavelength fluorescence measurement. Multiwavelength fluorescence imaging based on the VEcadherin within the aorta segment of a rat was achieved. The confocal microscopic system capable of fluorescence detection of cardiovascular tissue is a useful tool for measuring the biological properties in clinical applications.

&lt;i&gt;In Situ&lt;/i&gt; Study of Microstructural Development during Phase Transformation in Welding Process

The applications of laser scanning confocal microscopy (LSCM) system were introduced to the track the phase transformation of steel. For the low and high carbon steel, the microstructural change along various thermal cycles was directly observed with LSCM system. The nature of non-themoelastic martensite was directly observed. Furthermore, direct checking of the nucleation site of ferrite phase was carried out for aluminum killed weld metal and titanium killed-steel. It was carried out concerning to acicular ferrite formation in high strength and low alloy steel. The assessment of the pinning effect of alumiunu nitride on the austenite phase at high temperature was also carried out by using LSCM system.

Rapid confocal Raman imaging using a synchro multifoci-scan scheme for dynamic monitoring of single living cells

We developed a rapid multifoci-scan confocal Raman microscopy system for label-free molecular imaging of single living cells. A pair of galvo-mirrors were used to raster scan a single laser to generate multifoci excitations and another galvo-mirror synchronously projected Raman scattering from each foci onto a multichannel spectrograph such that multiple spectra were collected simultaneously. The image acquisition time is ∼40 times faster than in conventional point-scan Raman microscopy with diffraction-limited resolution retained. We demonstrated that this system can be used to monitor the germination dynamics of single bacterial spores with about 1.0 min resolution and 2.5 mW power at each focal point.

Near-IR fluorescence and reflectance confocal microscopy for imaging of quantum dots in mammalian skin

Understanding the skin penetration of nanoparticles (NPs) is an important concern due to the increasing presence of NPs in consumer products, including cosmetics. Technical challenges have slowed progress in evaluating skin barrier and NP factors that contribute to skin penetration risk. To limit sampling error and other problems associated with histological processing, many researchers are implementing whole tissue confocal or multiphoton microscopies. This work introduces a fluorescence and reflectance confocal microscopy system that utilizes near-IR excitation and emission to detect near-IR lead sulfide quantum dots (QDs) through ex vivo human epidermis. We provide a detailed prediction and experimental analysis of QD detection sensitivity and demonstrate detection of QD skin penetration in a barrier disrupted model. The unique properties of near-IR lead-based QDs will enable future studies that examine the impact of further barrier-disrupting agents on skin penetration of QDs and elucidate mechanistic insight into QD tissue interactions at the cellular level.