100 research outputs found
Scanning ion conductance microscopy: a convergent high-resolution technology for multi-parametric analysis of living cardiovascular cells
Cardiovascular diseases are complex pathologies that include alterations of various cell functions at the levels of intact tissue, single cells and subcellular signalling compartments. Conventional techniques to study these processes are extremely divergent and rely on a combination of individual methods, which usually provide spatially and temporally limited information on single parameters of interest. This review describes scanning ion conductance microscopy (SICM) as a novel versatile technique capable of simultaneously reporting various structural and functional parameters at nanometre resolution in living cardiovascular cells at the level of the whole tissue, single cells and at the subcellular level, to investigate the mechanisms of cardiovascular disease. SICM is a multimodal imaging technology that allows concurrent and dynamic analysis of membrane morphology and various functional parameters (cell volume, membrane potentials, cellular contraction, single ion-channel currents and some parameters of intracellular signalling) in intact living cardiovascular cells and tissues with nanometre resolution at different levels of organization (tissue, cellular and subcellular levels). Using this technique, we showed that at the tissue level, cell orientation in the inner and outer aortic arch distinguishes atheroprone and atheroprotected regions. At the cellular level, heart failure leads to a pronounced loss of T-tubules in cardiac myocytes accompanied by a reduction in Z-groove ratio. We also demonstrated the capability of SICM to measure the entire cell volume as an index of cellular hypertrophy. This method can be further combined with fluorescence to simultaneously measure cardiomyocyte contraction and intracellular calcium transients or to map subcellular localization of membrane receptors coupled to cyclic adenosine monophosphate production. The SICM pipette can be used for patch-clamp recordings of membrane potential and single channel currents. In conclusion, SICM provides a highly informative multimodal imaging platform for functional analysis of the mechanisms of cardiovascular diseases, which should facilitate identification of novel therapeutic strategies
Recommended from our members
Pellet imaging techniques on ASDEX
As part of a USDOE/ASDEX collaboration, a detailed examination of pellet ablation in ASDEX with a variety of diagnostics has allowed a better understanding of a number of features of hydrogen ice pellet ablation in a plasma. In particular, fast gated photos with an intensified Xybion CCD video camera allow in-situ velocity measurements of the pellet as it penetrates the plasma. With time resolution of typically 100 nanoseconds and exposures every 50 microseconds, the evolution of each pellet in a multi-pellet ASDEX tokamak plasma discharge can be followed. When the pellet cloud track has striations, the light intensity profile through the cloud is hollow (dark near the pellet), whereas at the beginning or near the end of the pellet trajectory the track is typically smooth (without striations) and has a gaussian-peaked light emission profile. New, single pellet Stark broadened D{sub {alpha}}D{sub {beta}}, and D{sub {gamma}} spectra, obtained with a tangentially viewing scanning mirror/spectrometer with Reticon array readout, are consistent with cloud densities of 2 {times} 10{sup 17}cm{sup {minus}3} or higher in the regions of strongest light emission. A spatially resolved array of D{sub {alpha}} detectors shows that the light variations during the pellet ablation are not caused solely by a modulation of the incoming energy flux as the pellet crosses rational q-surfaces, but instead are a result of a dynamic, non-stationary, ablation process. 20 refs., 4 figs
Nanoscale live-cell imaging using hopping probe ion conductance microscopy,
We describe hopping mode scanning ion conductance microscopy that allows noncontact imaging of the complex three-dimensional surfaces of live cells with resolution better than 20 nm. We tested the effectiveness of this technique by imaging networks of cultured rat hippocampal neurons and mechanosensory stereocilia of mouse cochlear hair cells. The technique allowed examination of nanoscale phenomena on the surface of live cells under physiological conditions. There is a great interest in developing methods to image live cells at nanoscale resolution. Scanning probe microscopy (SPM) is one approach to this problem and both atomic force microscopy (AFM) and scanning electrochemical microscopy (SECM) have been used to image live cells 1,2 . However, deformation of the soft and responsive cell by the AFM cantilever, particularly when imaging eukaryotic cells, represents a substantial problem for AFM. SECM, in contrast, involves no physical contact with the sample, but true topographic imaging of the convoluted surface of living cells with nanoscale resolution has not been reported. Scanning ion conductance microscopy (SICM) 3 is another form of SPM, which allows imaging of the cell surface under physiological conditions without physical contact and with a resolution of 3-6 nm 4,5 . Until now, SICM has been restricted to imaging relatively flat surfaces, as all other SPM techniques. This is because when the probe encounters a vertical structure, it inevitably collides with the specimen SICM is based on the phenomenon that the ion flow through a sharp fluid-filled nanopipette is partially occluded when the pipette approaches the surface of a cell 3 . In conventional SICM, a nanopipette is mounted on a three-dimensional piezoelectric translation stage and automatic feedback control moves the pipette up or down to keep the pipette current constant (the set point) while the sample is scanned in x and y directions. Thus, a pipette-sample separation, typically equal to the pipette's inner radius, is maintained during imaging. In hopping probe ion conductance microscopy (HPICM), we no longer use continuous feedback. Instead, at each imaging point, the pipette approaches the sample from a starting position that is above any of the surface features We illustrate the benefits of HPICM in In contrast to conventional raster scanning, HPICM has the additional advantage that the order of imaging pixels is not predetermined. Therefore, we divided the entire image into equal-sized square
Endocytic pathways: combined scanning ion conductance and surface confocal microscopy study
We introduce a novel high resolution scanning surface confocal microscopy technique that enables imaging of endocytic pits in apical membranes of live cells for the first time. The improved topographical resolution of the microscope together with simultaneous fluorescence confocal detection produces pairs of images of cell surfaces sufficient to identify single endocytic pits. Whilst the precise position and size of the pit is detected by the ion conductance microscope, the molecular nature of the pit, e.g. clathrin coated or caveolae, is determined by the corresponding green fluorescent protein fluorescence. Also, for the first time, we showed that flotillin 1 and 2 can be found co-localising with ~200-nm indentations in the cell membrane that supports involvement of this protein in endocytosis
VERITAS discovery of very high energy gamma-ray emission from S3 1227+25 and multiwavelength observations
We report the detection of very high energy gamma-ray emission from the
blazar S3 1227+25 (VER J1230+253) with the Very Energetic Radiation Imaging
Telescope Array System (VERITAS). VERITAS observations of the source were
triggered by the detection of a hard-spectrum GeV flare on May 15, 2015 with
the Fermi-Large Area Telescope (LAT). A combined five-hour VERITAS exposure on
May 16th and May 18th resulted in a strong 13 detection with a
differential photon spectral index, = 3.8 0.4, and a flux level
at 9% of the Crab Nebula above 120 GeV. This also triggered target of
opportunity observations with Swift, optical photometry, polarimetry and radio
measurements, also presented in this work, in addition to the VERITAS and
Fermi-LAT data. A temporal analysis of the gamma-ray flux during this period
finds evidence of a shortest variability timescale of = 6.2
0.9 hours, indicating emission from compact regions within the jet, and the
combined gamma-ray spectrum shows no strong evidence of a spectral cut-off. An
investigation into correlations between the multiwavelength observations found
evidence of optical and gamma-ray correlations, suggesting a single-zone model
of emission. Finally, the multiwavelength spectral energy distribution is well
described by a simple one-zone leptonic synchrotron self-Compton radiation
model.Comment: 18 pages, 6 figures. Accepted for publication in the Astrophysical
Journal (ApJ
Recommended from our members
Bright AGN Source List from the First Three Months of the Fermi Large Area Telescope All-Sky Survey
An Isolated Stellar-Mass Black Hole Detected Through Astrometric Microlensing
We report the first unambiguous detection and mass measurement of an isolated stellar-mass black hole (BH). We used the Hubble Space Telescope (HST) to carry out precise astrometry of the source star of the long-duration (t_E ~ 270 days), high-magnification microlensing event MOA-2011-BLG-191/OGLE-2011-BLG-0462, in the direction of the Galactic bulge. HST imaging, conducted at eight epochs over an interval of six years, reveals a clear relativistic astrometric deflection of the background star's apparent position. Ground-based photometry shows a parallactic signature of the effect of the Earth's motion on the microlensing light curve. Combining the HST astrometry with the ground-based light curve and the derived parallax, we obtain a lens mass of 7.1 +/- 1.3 M_Sun and a distance of 1.58 +/- 0.18 kpc. We show that the lens emits no detectable light, which, along with having a mass higher than is possible for a white dwarf or neutron star, confirms its BH nature. Our analysis also provides an absolute proper motion for the BH. The proper motion is offset from the mean motion of Galactic-disk stars at similar distances by an amount corresponding to a transverse space velocity of ~45 km/s, suggesting that the BH received a modest natal 'kick' from its supernova explosion. Previous mass determinations for stellar-mass BHs have come from radial-velocity measurements of Galactic X-ray binaries, and from gravitational radiation emitted by merging BHs in binary systems in external galaxies. Our mass measurement is the first ever for an isolated stellar-mass BH using any technique
Stretch-induced regional mechanoelectric dispersion and arrhythmia in the right ventricle of anesthetized lambs
- …