Defining a Superlens Operating Regime for Imaging Fluorescent Molecules

It has been shown that thin metal-based films can at certain frequencies act as planar near-field lenses for certain polarization components. A desirable property of such “lenses” is that they can also enhance and focus some large transverse spatial frequency components which contain sub-diffraction limit details. Over the last decade there has been much work in optimizing designs to reduce effects (such as material losses and surface roughness) that are detrimental to image reconstruction. One design that can reduce some of these undesirable effects, and which has received a fair amount of attention recently, is the stacked metal-dielectric superlens. Here we theoretically explore the imaging ability of such a design for the specific purpose of imaging a fluorescent dye (the common bio-marker GFP) in the vicinity of the superlens surface. Our calculations take into consideration the interaction (damping) of an oscillating electric dipole with the metallic layers in the superlens. We also assume a Gaussian frequency distribution spectrum for the dipole. We treat the metallic-alloy and dielectric-alloy layers separately using an appropriate effective medium theory. The transmission properties are evaluated via Transfer matrix (-matrix) calculations that were performed in the MatLab and MathCad environments. Our study shows that it is in principle possible to image fluorescent molecules using a simple bilayer planar superlens. We find that optimal parameters for such a superlens occur when the peak dipole emission-frequency is slightly offset from the Surface Plasmon resonance frequency of the metal-dielectric interfaces. The best resolution is obtained when the fluorescent molecules are not too close ( nm) or too far ( nm) from the superlens surface. The realization and application of a superlens with the specified design is possible using current nanofabrication techniques. When combined with e.g. a sub-wavelength grating structure (such as in the far-field superlens design previously proposed [1]) or a fast near-field scanning probe, it could provide a means for fast fluorescent imaging with sub-diffraction limit resolution

Social Entrepreneurship im deutschen Wohlfahrtsstaat: hybride Organisationen zwischen Markt, Staat und Gemeinschaft

Die Verfasser fragen nach der Bedeutung gesellschaftlichen Unternehmertums im etablierten deutschen System der Wohlfahrtsproduktion. Die Frage, ob gesellschaftliches Unternehmertum als Lückenbüßer oder Innovationsinkubatoren im sozialen Dienstleistungssektor fungieren, erfordert neben der Theoriebildung sowohl begriffliche Differenzierungen als auch empirische Überprüfungen. So müssen die organisationssoziologisch gesehen hybriden Sozialunternehmen von sozialen Unternehmen unterschieden werden. Am Beispiel der Bereiche Altenpflege und Kinder-/Jugendhilfe kann gezeigt werden, in wie fern der Sektor soziale Dienstleistungen zwar vom Typus der hybriden Sozialunternehmen geprägt ist, die empirischen Daten jedoch auch deutlich die Stabilität und Pfadabhängigkeit des bisherigen Wohlfahrtsmusters belegen. Mit ihrer Analyse der spezifischen Produktionsbedingungen des sozialen Dienstleistungssektors in Deutschland zeigen die Verfasser, dass eine Übertragung des angelsächsisch geprägten "social entrepreneurship"-Ansatzes auf die Realität des deutschen Wohlfahrtsstaates nur bedingt möglich ist, da Begrifflichkeiten und Konzepte an das jeweilige wohlfahrtsstaatliche Regime angepasst werden müssen und die jeweiligen institutionellen Konzepte zu berücksichtigen sind. (ICE2

Photounbinding of Calmodulin from a Family of CaM Binding Peptides

Background: Recent studies have shown that fluorescently labeled antibodies can be dissociated from their antigen by illumination with laser light. The mechanism responsible for the photounbinding effect, however, remains elusive. Here, we give important insights into the mechanism of photounbinding and show that the effect is not restricted to antibody/ antigen binding. Methodology/Principal Findings: We present studies of the photounbinding of labeled calmodulin (CaM) from a set of CaM-binding peptides with different affinities to CaM after one- and two-photon excitation. We found that the photounbinding effect becomes stronger with increasing binding affinity. Our observation that photounbinding can be influenced by using free radical scavengers, that it does not occur with either unlabeled protein or non-fluorescent quencher dyes, and that it becomes evident shortly after or with photobleaching suggest that photounbinding and photobleaching are closely linked. Conclusions/Significance: The experimental results exclude surface effects, or heating by laser irradiation as potential causes of photounbinding. Our data suggest that free radicals formed through photobleaching may cause a conformationa

Myocardial aging as a T-cell–mediated phenomenon

In recent years, the myocardium has been rediscovered under the lenses of immunology, and lymphocytes have been implicated in the pathogenesis of cardiomyopathies with different etiologies. Aging is an important risk factor for heart diseases, and it also has impact on the immune system. Thus, we sought to determine whether immunological activity would influence myocardial structure and function in elderly mice. Morphological, functional, and molecular analyses revealed that the age-related myocardial impairment occurs in parallel with shifts in the composition of tissue-resident leukocytes and with an accumulation of activated CD4+ Foxp3- (forkhead box P3) IFN-γ+ T cells in the heart-draining lymph nodes. A comprehensive characterization of different aged immune-deficient mouse strains revealed that T cells significantly contribute to age-related myocardial inflammation and functional decline. Upon adoptive cell transfer, the T cells isolated from the mediastinal lymph node (med-LN) of aged animals exhibited increased cardiotropism, compared with cells purified from young donors or from other irrelevant sites. Nevertheless, these cells caused rather mild effects on cardiac functionality, indicating that myocardial aging might stem from a combination of intrinsic and extrinsic (immunological) factors. Taken together, the data herein presented indicate that heart-directed immune responses may spontaneously arise in the elderly, even in the absence of a clear tissue damage or concomitant infection. These observations might shed new light on the emerging role of T cells in myocardial diseases, which primarily affect the elderly population.info:eu-repo/semantics/publishedVersio

Dimerization of melanocortin 4 receptor controls puberty onset and body size polymorphism

Xiphophorus fish exhibit a clear phenotypic polymorphism in puberty onset and reproductive strategies of males. In X. nigrensis and X. multilineatus, puberty onset is genetically determined and linked to a melanocortin 4 receptor (Mc4r) polymorphism of wild-type and mutant alleles on the sex chromosomes. We hypothesized that Mc4r mutant alleles act on wild-type alleles by a dominant negative effect through receptor dimerization, leading to differential intracellular signaling and effector gene activation. Depending on signaling strength, the onset of puberty either occurs early or is delayed. Here, we show by Förster Resonance Energy Transfer (FRET) that wild-type Xiphophorus Mc4r monomers can form homodimers, but also heterodimers with mutant receptors resulting in compromised signaling which explains the reduced Mc4r signaling in large males. Thus, hetero- vs. homo- dimerization seems to be the key molecular mechanism for the polymorphism in puberty onset and body size in male fish

Sharpening emitter localization in front of a tuned mirror

Single-molecule localization microscopy (SMLM) aims for maximized precision and a high signal-to-noise ratio1. Both features can be provided by placing the emitter in front of a metal-dielectric nanocoating that acts as a tunedmirror2–4. Here, we demonstrate that a higher photon yield at a lower background on biocompatible metal-dielectric nanocoatings substantially improves SMLM performance and increases the localization precision by up to a factor oftwo. The resolution improvement relies solely on easy-to-fabricate nanocoatings on standard glass coverslips and is spectrally and spatially tunable by the layer design and wavelength, as experimentally demonstrated for dual-color SMLM in cells.Publisher PDFPeer reviewe

Intravital imaging of megakaryocytes

The dynamics of platelet formation could only be investigated since the development of two-photon microscopy in combination with suitable fluorescent labeling strategies. In this review paper, we give an overview of recent advances in fluorescence imaging of the bone marrow that have contributed to our understanding of platelet biogenesis during the last decade. We make a brief survey through the perspectives and limitations of today’s intravital imaging, but also discuss complementary methods that may help to piece together the puzzle of megakaryopoiesis and platelet formation

Simultaneous two-photon excitation of distinct labels for dual-color fluorescence crosscorrelation analysis

Confocal fluorescence correlation spectroscopy as a time-averaging fluctuation analysis combining maximum sensitivity with high statistical confidence has proved to be a very versatile and powerful tool for detection and temporal investigation of biomolecules at ultralow concentrations on surfaces, in solutions, and in living cells. To probe the interaction of different molecular species for a detailed understanding of biologically relevant mechanisms, crosscorrelation studies on dual or multiple fluorophore assays with spectrally distinct excitation and emission are particularly promising. Despite the considerable improvement of detection specificity provided by fluorescence crosscorrelation analysis, few applications have so far been reported, presumably because of the practical challenges of properly aligning and controlling the stability of the experimental setup. In this work, we demonstrate that two-photon excitation combined with dual-color fluorescence correlation spectroscopy can be the key to simplifying simultaneous investigations of multiple fluorescent species significantly on a single-molecule scale. Two-photon excitation allows accession of common fluorophores of largely distinct emission by the same excitation wavelength, because differences in selection rules and vibronic coupling can induce considerable shifts between the one-photon and two-photon excitation spectra. The concept of dual-color two-photon fluorescence crosscorrelation analysis is introduced and experimentally demonstrated with an established assay probing the selective cleavage of dual-labeled DNA substrates by restriction endonuclease EcoRI