Cornsweet surfaces for selective contrast enhancement

A typical goal when enhancing the contrast of images is to increase the perceived contrast without altering the original feel of the image. Such contrast enhancement can be achieved by modelling Cornsweet profiles into the image. We demonstrate that previous methods aiming to model Cornsweet profiles for contrast enhancement, often employing the unsharp mask operator, are not robust to image content. To achieve robustness, we propose a fundamentally di erent vector-centric approach with Cornsweet surfaces. Cornsweet surfaces are parametrised 3D surfaces (2D in space, 1D in luminance enhancement) that are extruded or depressed in the luminance dimension to create countershading that respects image structure. In contrast to previous methods, our method is robust against the topology of the edges to be enhanced and the relative luminance across those edges. In user trials, our solution was significantly preferred over the most related contrast enhancement method.Kosinka was funded by EPSRC grant EP/H024816/1. Lieng was funded by a scholarship from the Norwegian Government.This is the accepted manuscript. The final version is available from Elsevier at http://www.sciencedirect.com/science/article/pii/S0097849314000405

Imaging Ca2+ concentration changes at the secretory vesicle surface with a recombinant targeted cameleon

AbstractRegulated exocytosis involves the Ca2+-triggered fusion of secretory vesicles with the plasma membrane, by activation of vesicle membrane Ca2+-binding proteins [1]. The Ca2+-binding sites of these proteins are likely to lie within 30 nm of the vesicle surface, a domain in which changes in Ca2+ concentration cannot be resolved by conventional fluorescence microscopy. A fluorescent indicator for Ca2+ called a yellow ‘cameleon’ (Ycam2) – comprising a fusion between a cyan-emitting mutant of the green fluorescent protein (GFP), calmodulin, the calmodulin-binding peptide M13 and an enhanced yellow-emitting GFP – which is targetable to specific intracellular locations, has been described [2]. Here, we generated a fusion between phogrin, a protein that is localised to secretory granule membranes [3], and Ycam2 (phogrin–Ycam2) to monitor changes in Ca2+ concentration ([Ca2+]) at the secretory vesicle surface ([Ca2+]gd) through alterations in fluorescence resonance energy transfer (FRET) between the linked cyan and yellow fluorescent proteins (CFP and YFP, respectively) in Ycam2. In both neuroendocrine PC12 and MIN6 pancreatic β cells, apparent resting values of cytosolic [Ca2+] and [Ca2+]gd were similar throughout the cell. In MIN6 cells following the activation of Ca2+ influx, the minority of vesicles that were within ∼1 μm of the plasma membrane underwent increases in [Ca2+]gd that were significantly greater than those experienced by deeper vesicles, and greater than the apparent cytosolic [Ca2+] change. The ability to image both global and compartmentalised [Ca2+] changes with recombinant targeted cameleons should extend the usefulness of these new Ca2+ probes

Molecular genetic features and risk assessment in a series of 30 patients who underwent an operation for gastrointestinal stromal tumours

Background: The objective of the study was to investigate the relationship between molecular genetic features and the standard criteria of risk assessment in patients affected by gastrointestinal stromal tumours (GISTs). Methods: A review was conducted of a series of 30 patients, with a mean age of 67 years, who underwent surgery for primary GISTs. R0 resection was accomplished in 27 patients. CD117, CD34 desmin, vimentin, S-100 and smooth muscle actin were immunohistochemically tested to achieve a diagnosis of GIST. The loss of wild-type KIT or platelet-derived growth factor receptor alpha (PDGFRα) genes was investigated by sequencing the tumour DNA. Results: Tumour genes mutations were reported in 23 patients (77%), and wild-type in seven. Mutations on the KIT gene occurred in 18 patients, and mutations on the PDGFRα gene in five. The average sizes of the GIST were 8.7 cm, 5.4 cm and 5.9 cm for KIT gene-mutated, PDGFRα gene-mutated and wild-type tumours, respectively. KIT gene mutations were detected in 50% of gastric and in 70% of extragastric GISTs. Moreover, 70% of tumours with a mitotic rate ≥ 5 x 50 highpower fields (HPFs) underwent KIT gene mutations. Conversely, PDGFRα mutations were observed only in gastric GISTs with a mitotic rate ≤ 5 x 50 HPFs. By stratifying GISTs according to classes of risk, KIT mutation was shown in most of the high-risk tumours. PDGFRα mutations occurred exclusively in lower classes of risk. Conclusion: Molecular analysis data might have a role as a prognostic variable in models of risk assessment for patients with GISTs

Glucose generates sub-plasma membrane ATP microdomains in single islet beta-cells. Potential role for strategically located mitochondria.

Abstract Increases in the concentration of free ATP within the islet β-cell may couple elevations in blood glucose to insulin release by closing ATP-sensitive K+(KATP) channels and activating Ca2+ influx. Here, we use recombinant targeted luciferases and photon counting imaging to monitor changes in free [ATP] in subdomains of single living MIN6 and primary β-cells. Resting [ATP] in the cytosol ([ATP]c), in the mitochondrial matrix ([ATP]m), and beneath the plasma membrane ([ATP]pm) were similar (∼1 mm). Elevations in extracellular glucose concentration (3–30 mm) increased free [ATP] in each domain with distinct kinetics. Thus, sustained increases in [ATP]m and [ATP]pm were observed, but only a transient increase in [ATP]c. However, detectable increases in [ATP]c and [ATP]pm, but not [ATP]m, required extracellular Ca2+. Enhancement of glucose-induced Ca2+ influx with high [K+] had little effect on the apparent [ATP]c and [ATP]m increases but augmented the [ATP]pm increase. Underlying these changes, glucose increased the mitochondrial proton motive force, an effect mimicked by high [K+]. These data support a model in which glucose increases [ATP]m both through enhanced substrate supply and by progressive Ca2+-dependent activation of mitochondrial enzymes. This may then lead to a privileged elevation of [ATP]pm, which may be essential for the sustained closure of KATP channels. Luciferase imaging would appear to be a useful new tool for dynamic in vivo imaging of free ATP concentration