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The combination of lineage tracing and immunohistochemistry has helped to identify subpopulations and fate of hepatic stellate cells (HSC) in murine liver. HSC are sinusoidal pericytes that act as myofibroblast precursors after liver injury. Single cell RNA sequencing approaches have recently helped to differentiate central and portal HSC. A specific Cre line to lineage trace portal HSC has not yet been described. We used three Cre lines (Lrat-Cre, PDGFRß-CreERT2 and SMMHC-CreERT2) known to label mesenchymal cells including HSC in combination with a tdTomato-expressing reporter. All three Cre lines labeled populations of HSC as well as smooth muscle cells (SMC). Using the SMMHC-CreERT2, we identified a subtype of HSC in the periportal area of the hepatic lobule (termed zone 1-HSC). We lineage traced tdTomato-expressing zone 1-HSC over 1 year, described fibrotic behavior in two fibrosis models and investigated their possible role during fibrosis. This HSC subtype resides in zone 1 under healthy conditions; however, zonation is disrupted in preclinical models of liver fibrosis (CCl4 and MASH). Zone 1-HSC do not transform into αSMA-expressing myofibroblasts. Rather, they participate in sinusoidal capillarization. We describe a novel subtype of HSC restricted to zone 1 under physiological conditions and its possible function after liver injury. In contrast to the accepted notion, this HSC subtype does not transform into αSMA-positive myofibroblasts; rather, zone 1-HSC adopt properties of capillary pericytes, thereby participating in sinusoidal capillarization.
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Células Estrelladas Hepáticas , Cirrosis Hepática , Miofibroblastos , Animales , Células Estrelladas Hepáticas/metabolismo , Células Estrelladas Hepáticas/patología , Miofibroblastos/metabolismo , Miofibroblastos/patología , Ratones , Cirrosis Hepática/patología , Cirrosis Hepática/metabolismo , Hígado/patología , Hígado/metabolismo , Pericitos/metabolismo , Pericitos/patología , Linaje de la Célula , Masculino , Diferenciación Celular , Modelos Animales de Enfermedad , Ratones Endogámicos C57BLRESUMEN
Resonant enhancement inside an optical cavity has been a wide-spread approach to increase efficiency of nonlinear optical conversion processes while reducing the demands on the driving laser power. This concept has been particularly important for high harmonic generation XUV sources, where passive femtosecond enhancement cavities allowed significant increase in repetition rates required for applications in photoelectron spectroscopy, XUV frequency comb spectroscopy, including the recent endeavor of thorium nuclear clock development. In addition to passive cavities, it has been shown that comparable driving conditions can be achieved inside mode-locked thin-disk laser oscillators, offering a simplified single-stage alternative. This approach is less sensitive to losses thanks to the presence of gain inside the cavity and should thus allow higher conversion efficiencies through tolerating higher intensity in the gas target. Here, we show that the intra-oscillator approach can indeed surpass the much more mature technology of passive enhancement cavities in terms of XUV flux, even reaching comparable values to single-pass sources based on chirped-pulse fiber amplifier lasers. Our system operates at 17 MHz repetition rate generating photon energies between 60 eV and 100 eV. Importantly, this covers the highly attractive wavelength for the silicon industry of 13.5 nm at which our source delivers 60 nW of outcoupled average power per harmonic order.
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Recombinant adenoviruses are widely used in clinical and laboratory applications. Despite the wide variety of available sero- and genotypes, only a fraction is utilized in vivo. As adenoviruses are a large group of viruses, displaying many different tropisms, immune epitopes, and replication characteristics, the merits of translating these natural benefits into vector applications are apparent. This translation, however, proves difficult, since while research has investigated the application of these viruses, there are no universally applicable rules in vector design for non-classical adenovirus types. In this paper, we describe a generalized workflow that allows vectorization, rescue, and cloning of all adenoviral species to enable the rapid development of new vector variants. We show this using human and simian adenoviruses, further modifying a selection of them to investigate their gene transfer potential and build potential vector candidates for future applications.
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Vectores Genéticos , Recombinación Genética , Vectores Genéticos/genética , Humanos , Adenoviridae/genética , Adenovirus Humanos/genética , Animales , Técnicas de Transferencia de Gen , Adenovirus de los Simios/genética , Clonación Molecular/métodosRESUMEN
HYPOTHESIS: Amphiphilic diblock copolymers are known to increase the surfactant's efficiency to stabilize microemulsion, leading to higher structural order and monolayer rigidity. We thus seek to evaluate whether the addition of such polymers alters the shear behavior of bicontinuous microemulsions, in particular, their shear transformation towards lamellar structures. EXPERIMENTS: We examine the initial structure and shear response of bicontinuous /n-octane//PEP5-b-PEO5 microemulsions by coupling microfluidics with small-angle neutron scattering (SANS), attaining wall shear rates in excess of . The azimuthal analysis of the obtained 2D scattering patterns allows us to follow their structural transformation by means of the degree of anisotropy. FINDINGS: The amphiphilic diblock copolymer promotes the shear-induced transformation of bicontinuous microemulsions, resulting in up to â¼ higher degrees of anisotropy than for corresponding polymer-free microemulsions. The increased shear response observed with increasing polymer content is rationalized by combining the influence of domain size and viscosity with the stability limits of the bicontinuous microemulsion in the isothermal phase diagram. As a result, a consistent description of the degree of anisotropy is obtained, enabling the prediction of the shear-induced bicontinuous-to-lamellar transformation.
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While recombinant adenoviruses (rAds) are widely used in both laboratory and medical gene transfer, library-based applications using this vector platform are not readily available. Recently, we developed a new method, the CRISPR-Cas9 mediated in vivo terminal resolution aiding high-efficiency rescue of rAds from recombinant DNA. Here we report on a genetic workflow that allows construction of bacterial artificial chromosome-based rAd libraries reconstituted using highly efficient terminal resolution. We utilized frequent, pre-existing genomic sequences to allow the insertion of a selection marker, complementing two selected target sites into novel endonuclease recognition sites. In the second step, this selection marker is replaced with a transgene or mutation of interest via Gibson assembly. Our approach does not cause unwanted genomic off-target mutations while providing substantial flexibility for the site and nature of the genetic modification. This new genetic workflow, which we termed half site-directed fragment replacement (HFR) allows the introduction of more than 106 unique modifications into rAd encoding BACs using laboratory scale methodology. To demonstrate the power of HFR, we rescued barcoded viral vector libraries yielding a diversity of approximately 2.5 × 104 unique rAds per cm2 of transfected cell culture.
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There is a rising number in complications associated with more cardiac electrical devices implanted (CIED). Infection and lead dysfunction are reasons to perform transvenous lead extraction. An ideal anaesthetic approach has not been described yet. Most centres use general anaesthesia, but there is a lack in studies looking into deep sedation (DS) as an anaesthetic approach. We report our retrospective experience for a large number of procedures performed with deep sedation as a primary approach. Extraction procedures performed between 2011 and 2018 in our electrophysiology laboratory have been included retrospectively. We began by applying a bolus injection of piritramide followed by midazolam as primary medication and would add etomidate if necessary. For extraction of leads a stepwise approach with careful traction, locking stylets, dilator sheaths, mechanical rotating sheaths and if needed snares and baskets has been used. A total of 780 leads in 463 patients (age 69.9 ± 12.3, 31.3% female) were extracted. Deep sedation was successful in 97.8% of patients. Piritramide was used as the main analgesic medication (98.5%) and midazolam as the main sedative (94.2%). Additional etomidate was administered in 15.1% of cases. In 2.2% of patients a conversion to general anaesthesia was required as adequate level of DS was not achieved before starting the procedure. Sedation related complications occurred in 1.1% (n = 5) of patients without sequalae. Deep sedation with piritramide, midazolam and if needed additional etomidate is a safe and feasible strategy for transvenous lead extraction.
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Anestésicos , Sedación Profunda , Desfibriladores Implantables , Etomidato , Marcapaso Artificial , Humanos , Femenino , Masculino , Midazolam/efectos adversos , Estudios Retrospectivos , Pirinitramida , Sedación Profunda/efectos adversos , Sedación Profunda/métodos , Remoción de Dispositivos/efectos adversos , Remoción de Dispositivos/métodos , Resultado del Tratamiento , Marcapaso Artificial/efectos adversosRESUMEN
BACKGROUND: Infection, lead dysfunction and system upgrades are all reasons that transvenous lead extraction is being performed more frequently. Many centres focus on a single method for lead extraction, which can lead to either lower success rates or higher rates of major complications. We report our experience with a systematic approach from a less invasive to a more invasive strategy without the use of laser sheaths. METHODS: Consecutive extraction procedures performed over a period of seven years in our electrophysiology laboratory were included. We performed a stepwise approach with careful traction, lead locking stylets (LLD), mechanical non-powered dilator sheaths, mechanical powered sheaths and, if needed, femoral snares. RESULTS: In 463 patients (age 69.9 ± 12.3, 31.3% female) a total of 780 leads (244 ICD leads) with a mean lead dwelling time of 5.4 ± 4.9 years were identified for extraction. Success rates for simple traction, LLD, mechanical non-powered sheaths and mechanical powered sheaths were 31.5%, 42.7%, 84.1% and 92.6%, respectively. A snare was used for 40 cases (as the primary approach for 38 as the lead structure was not intact and stepwise approach was not feasible) and was successful for 36 leads (90.0% success rate). Total success rate was 93.1%, clinical success rate was 94.1%. Rate for procedural failure was 1.1%. Success for less invasive steps and overall success for extraction was associated with shorter lead dwelling time (p < 0.001). Major procedure associated complications occurred in two patients (0.4%), including one death (0.2%). A total of 36 minor procedure-associated complications occurred in 30 patients (6.5%). Pocket hematoma correlated significantly with uninterrupted dual antiplatelet therapy (p = 0.001). Pericardial effusion without need for intervention was associated with long lead dwelling time (p = 0.01) and uninterrupted acetylsalicylic acid (p < 0.05). CONCLUSION: A stepwise approach with a progressive invasive strategy is effective and safe for transvenous lead extraction.
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The Dean-Kawasaki equation-a strongly singular SPDE-is a basic equation of fluctuating hydrodynamics; it has been proposed in the physics literature to describe the fluctuations of the density of N independent diffusing particles in the regime of large particle numbers Nâ«1. The singular nature of the Dean-Kawasaki equation presents a substantial challenge for both its analysis and its rigorous mathematical justification. Besides being non-renormalisable by the theory of regularity structures by Hairer et al., it has recently been shown to not even admit nontrivial martingale solutions. In the present work, we give a rigorous and fully quantitative justification of the Dean-Kawasaki equation by considering the natural regularisation provided by standard numerical discretisations: We show that structure-preserving discretisations of the Dean-Kawasaki equation may approximate the density fluctuations of N non-interacting diffusing particles to arbitrary order in N-1 (in suitable weak metrics). In other words, the Dean-Kawasaki equation may be interpreted as a "recipe" for accurate and efficient numerical simulations of the density fluctuations of independent diffusing particles.
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Coupling microfluidics and small-angle neutron scattering (SANS), we investigate the influence of shear flow on a model bicontinuous microemulsion of D2O/n-octane/C10E4, examining the role of membrane volume fraction in the transformation towards a lamellar structure. We employ a contraction-expansion geometry with flow velocities in excess of 10 m s-1 and spatially map the microfluidic field using a small SANS beam, illuminating down to 10 nL sample volumes. The shear-induced, progressive, bicontinuous-to-lamellar transition is found to be promoted by additional extensional flow (>103 s-1), while fast relaxation kinetics (<2 ms) return the scattering pattern to isotropic shortly after the constriction. Further, increasing the domain size of the bicontinuous structure (determined by the membrane volume fraction) appears to amplify its response to shear. Hence, the structural changes within the dilute bicontinuous microemulsions simply scale with the volume fraction of the membrane. By contrast, the stronger response of the microemulsion with the smallest domain size, located near the bicontinuous/lamellar coexistence, indicates an influence of an already more ordered structure with fewer passages. Our findings provide insight into the high shear behaviour of microemulsions of both academic and industrial relevance.
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HYPOTHESIS: Shear flow applied to bicontinuous microemulsions is expected to induce a transition to lamellae via the suppression of surfactant monolayer fluctuations. Compared to the topologically analogous L3 (sponge) phase, composed of surfactant bilayers, this transition is likely to occur at much higher shear rates. EXPERIMENTS: We examine the flow response of a model bicontinuous microemulsion, D2O/n-octane/C10E4 by coupling microfluidics with small-angle neutron scattering (SANS), attaining wall shear rates in excess of 105 s-1. The reduction of probed sample volumes down to â¼10 nL allows the spatial mapping of the structural and orientation changes within the microchannel, as a function of the flow field components. FINDINGS: With increasing flow rate, we observe a gradual increase in scattering anisotropy, accompanied by a decrease of the microemulsion domain size along the main flow orientation. A consistent description of the degree of anisotropy was obtained when considering the velocity gradient along the scattering plane perpendicular to the flow. We discuss the flow dependence of the effective bending rigidity, rationalizing a strong influence of shear on thermal membrane fluctuations. Assuming a similar shear dependence for the saddle splay modulus, the bicontinuous-to-lamellar transition can be attributed to the gradual disappearance of inter-lamellar passages.
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We experimentally demonstrate an efficient and broadband extreme-ultraviolet light (XUV) out-coupling mechanism of intra-cavity generated high harmonics. The mechanism is based on a coated grazing-incidence plate (GIP), which utilizes the enhanced reflectivity of s-polarized light in comparison to p-polarized light for large angles of incidence (AoI). We design and produce a 60°-AoI coated GIP, tailored specifically for the high demands inside a sub-50-fs Kerr-lens mode-locked Yb:YAG thin-disk laser oscillator in which high harmonic generation (HHG) is driven at â¼450 MW peak power and 17 MHz repetition rate. The coated GIP features an XUV out-coupling efficiency of >25% for photon energies ranging from 10 eV to 60 eV while being anti-reflective for the driving laser field. The XUV spectra reach up to 52 eV in argon and 30 eV in xenon. In a single harmonic, we out-couple 1.3 µW of XUV average power at 37 eV in argon and 5.4 µW at 25 eV in xenon. The combination of an improved HHG driving laser performance and the out-coupling via the coated GIP enabled us to increase the out-coupled XUV average power in a single harmonic by a factor of 20 compared to previous HHG inside ultrafast laser oscillators. Our source approaches the state-of-the-art out-coupled XUV power levels per harmonic of femtosecond enhancement cavities operating at comparable photon energies.
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Recombinant adenovirus (rAd) vectors represent one of the most frequently used vehicles for gene transfer applications in vitro and in vivo. rAd genomes are constructed in Escherichia coli where their genomes can be maintained, propagated, and modified in form of circular plasmids or bacterial artificial chromosomes. Although the rescue of rAds from their circular plasmid or bacmid forms is well established, it works with relatively low primary efficiency, preventing this technology for library applications. To overcome this barrier, we tested a novel strategy for the reconstitution of rAds that utilizes the CRISPR/Cas-machinery to cleave the circular rAd genomes in close proximity to their inverted terminal repeats (ITRs) within the producer cells upon transfection. This CRISPR/Cas-mediated in vivo terminal resolution allowed efficient rescue of vectors derived from different human adenovirus (HAdV) species. By this means, it was not only possible to increase the efficiency of virus rescue by about 50-fold, but the presented methodology appeared also remarkably simpler and faster than traditional rAd reconstitution methods.
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Facing an ongoing organ shortage in transplant medicine, strategies to increase the use of organs from marginal donors by objective organ assessment are being fostered. In this context, normothermic machine perfusion provides a platform for ex vivo organ evaluation during preservation. Consequently, analytical tools are emerging to determine organ quality. In this study, hyperspectral imaging (HSI) in the wavelength range of 550-995 nm was applied. Classification of 26 kidneys based on HSI was established using KidneyResNet, a convolutional neural network (CNN) based on the ResNet-18 architecture, to predict inulin clearance behavior. HSI preprocessing steps were implemented, including automated region of interest (ROI) selection, before executing the KidneyResNet algorithm. Training parameters and augmentation methods were investigated concerning their influence on the prediction. When classifying individual ROIs, the optimized KidneyResNet model achieved 84% and 62% accuracy in the validation and test set, respectively. With a majority decision on all ROIs of a kidney, the accuracy increased to 96% (validation set) and 100% (test set). These results demonstrate the feasibility of HSI in combination with KidneyResNet for non-invasive prediction of ex vivo kidney function. This knowledge of preoperative renal quality may support the organ acceptance decision.
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We experimentally investigate the limits of pulse duration in a Kerr-lens mode-locked Yb:YAG thin-disk laser (TDL) oscillator. Thanks to its excellent mechanical and optical properties, Yb:YAG is one of the most used gain materials for continuous-wave and pulsed TDLs. In mode-locked operation, its 8-nm wide gain bandwidth only directly supports pulses with a minimum duration of approximately 140 fs. For achieving shorter pulses, a Kerr-lens mode-locked TDL oscillator can be operated in the strongly self-phase modulation (SPM) broadened regime. Here, the spectral bandwidth of the oscillating pulse exceeds the available gain bandwidth by generating additional frequencies via SPM inside the Kerr medium. In this work, we study and compare different laser configurations in the strongly SPM-broadened regime. Starting with a configuration providing 84-fs pulses at 69 W average power at 17 MHz repetition rate, we reduce the pulse duration by optimizing various mode-locking parameters. One crucial parameter is the dispersion control which was provided by in-house-developed dispersive mirrors produced by ion-beam sputtering (IBS). We discuss trade-offs in average power, pulse duration, efficiency, and intra-cavity peak power. For the configuration operating at the highest SPM-broadening, we achieve a minimum pulse duration of 27 fs, which represents the shortest pulse duration directly generated by any ultrafast TDL oscillator. The corresponding full width at half maximum (FWHM) spectral bandwidth exceeds more than five times the FWHM gain bandwidth. The average output power of 3.3 W is moderate for ultrafast TDL oscillators, but higher than other Yb-based laser oscillators operating at this pulse duration. Additionally, the corresponding intra-cavity peak power of 0.8 GW is highly attractive for implementing intra-cavity extreme nonlinear optical interactions such as high harmonic generation.
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Assessing species status and making classification decisions under the Endangered Species Act is a critical step towards effective species conservation. However, classification decisions are liable to two errors: i) failing to classify a species as threatened or endangered that should be classified (underprotection), or ii) classifying a species as threatened or endangered when it is not warranted (overprotection). Recent surveys indicate threatened spectacled eider populations are increasing in western Alaska, prompting the U.S. Fish and Wildlife Service to reconsider the federal listing status. There are multiple criteria set for assessing spectacled eider status, and here we focus on the abundance and decision analysis criteria. We estimated population metrics using state-space models for Alaskan breeding populations of spectacled eiders. We projected abundance over 50 years using posterior estimates of abundance and process variation to estimate the probability of quasi-extinction. The decision analysis maps the risk of quasi-extinction to the loss associated with making a misclassification error (i.e., underprotection) through a loss function. Our results indicate that the Yukon Kuskokwim Delta breeding population in western Alaska has met the recovery criteria but the Arctic Coastal Plain population in northern Alaska has not. The methods employed here provide an example of accounting for uncertainty and incorporating value judgements in such a way that the decision-makers may understand the risk of committing a misclassification error. Incorporating the abundance threshold and decision analysis in the reclassification criteria greatly increases the transparency and defensibility of the classification decision, a critical aspect for making effective decisions about species management and conservation.
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Toma de Decisiones en la Organización , Técnicas de Apoyo para la Decisión , Patos , Especies en Peligro de Extinción/legislación & jurisprudencia , Alaska , Animales , Teorema de Bayes , Seguimiento de Parámetros Ecológicos/estadística & datos numéricos , Especies en Peligro de Extinción/estadística & datos numéricos , Estados Unidos , El YukónRESUMEN
Free-running dual-comb systems based on a single laser cavity are an attractive next generation technology for a wide variety of applications. The high average power achievable by dual-comb thin-disk laser (TDL) oscillators make this technology especially attractive for spectroscopy and sensing applications in the molecular fingerprint region enabled by nonlinear frequency conversion. However, the high noise levels of TDL oscillators, e.g., induced by the turbulent water-cooling of the disk, are a severe challenge for spectroscopic applications. In this contribution, we confirm for the first time the suitability of dual-comb TDLs for high-resolution spectroscopy. Based on the novel concept of polarization splitting inside a TDL, our oscillator generates two asynchronous pulse trains of 240-fs pulse duration at 6-W and 8-W average power per pulse train and â¼97-MHz repetition rate at a central wavelength of 1030 nm. In the first detailed noise investigation of such a system, we identify the repetition frequency as the dominant noise term and show that â¼85% of the frequency noise of the comb lines of both pulse trains is correlated (integrated from 200 Hz to 20 kHz). We detect the absorption spectrum of acetylene in free-running operation within a measurement time of 1 millisecond. Being highly suitable for nonlinear frequency conversion, we believe the here presented result is an important step towards simple yet powerful mid-infrared dual-comb systems for high-resolution spectroscopy.
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We demonstrate that Kerr lens modelocking is well-suited for operating an ultrafast thin-disk laser with intra-oscillator high harmonic generation (HHG) in the 100-fs pulse duration regime. Exploiting nearly the full emission bandwidth of the gain material Yb:YAG, we generate 105-fs pulses with an intracavity peak power of 365 MW and an intracavity average power of 470 W. We drive HHG in argon with a peak intensity of â¼7â 1013 W/cm2 at a repetition rate of 11 MHz. Extreme-ultraviolet (XUV) light is generated up to the 31st harmonic order (H31) at 37 eV, with an average power of â¼0.4 µW in H25 at 30 eV. This work presents a considerable increase in performance of XUV sources based on intra-oscillator HHG and confirms that this approach is a promising technology for simple and portable XUV sources at MHz repetition rates.
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OBJECTIVE: This study provides a comprehensive overview of the associations of five adipokines (adiponectin, chemerin, galectin-3, leptin, and resistin) with fat deposits, behavioral risk factors, and metabolic phenotypes. METHODS: Using multivariable linear and logistic regression models, cross-sectional data from 4,116 participants of the population-based Study of Health in Pomerania were analyzed. RESULTS: Participants with obesity showed higher chemerin, galectin-3, and leptin but showed lower adiponectin concentrations. Independently of other fat compounds, liver fat content, visceral adipose tissue, and subcutaneous adipose tissue (SAT) were inversely associated with adiponectin. Independent positive associations of liver fat content and SAT with chemerin as well as of SAT with galectin-3 and leptin were observed. Physically inactive participants had higher chemerin and leptin concentrations. Smokers had higher chemerin and galectin-3 as well as lower leptin. Alcohol consumption was associated with adiponectin (positive) and resistin (inverse). All adipokines were associated with at least one lipid marker. Associations with glucose metabolism were seen for adiponectin, chemerin, galectin-3, and leptin. CONCLUSIONS: High adiponectin concentrations were related to favorable metabolic conditions, whereas high chemerin, galectin-3, and leptin were associated with an unfavorable metabolic profile. High leptin seems to be primarily indicative of obesity, whereas high adiponectin and chemerin are associated with a broader range of metabolic phenotypes.
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Adipoquinas/metabolismo , Biomarcadores/metabolismo , Metabolómica/métodos , Obesidad/metabolismo , Adulto , Estudios Transversales , Femenino , Humanos , Masculino , Persona de Mediana Edad , Fenotipo , Factores de RiesgoRESUMEN
We investigate power-scaling of a Kerr lens mode-locked (KLM) Yb:Lu2O3 thin-disk laser (TDL) oscillator operating in the sub-100-fs pulse duration regime. Employing a scheme with higher round-trip gain by increasing the number of passes through the thin-disk gain element, we increase the average power by a factor of two and the optical-to-optical efficiency by a factor of almost three compared to our previous sub-100-fs mode-locking results. The oscillator generates pulses with a duration of 95 fs at 21.1 W average power and 47.9 MHz repetition rate. We discuss the cavity design for continuous-wave and mode-locked operation and the estimation of the focal length of the Kerr lens. Unlike to usual KLM TDL oscillators, an operation at the edge of the stability zone in continuous-wave operation is not required. This work shows that KLM TDL oscillators based on the gain material Yb:Lu2O3 are an excellent choice for power-scaling of laser oscillators in the sub-100-fs regime, and we expect that such lasers will soon operate at power levels in excess of hundred watts.
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We report on the condensation of microcavity exciton polaritons under optical excitation in a microcavity with four embedded InGaAs quantum wells. The polariton laser is characterized by a distinct non-linearity in the input-output-characteristics, which is accompanied by a drop of the emission linewidth indicating temporal coherence and a characteristic persisting emission blueshift with increased particle density. The temporal coherence of the device at threshold is underlined by a characteristic drop of the second order coherence function to a value close to 1. Furthermore an external electric field is used to switch between polariton regime, polariton condensate and photon lasing.