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Bone healing involves the interplay of immune cells, mesenchymal cells, and vasculature over the time course of regeneration. Approaches to quantify the spatiotemporal aspects of bone healing at cellular resolution during long bone healing do not yet exist. Here, a novel technique termed Limbostomy is presented, which combines intravital microendoscopy with an osteotomy. This design allows a modular combination of an internal fixator plate with a gradient refractive index (GRIN) lens at various depths in the bone marrow and can be combined with a surgical osteotomy procedure. The field of view (FOV) covers a significant area of the fracture gap and allows monitoring cellular processes in vivo. The GRIN lens causes intrinsic optical aberrations which have to be corrected. The optical system was characterized and a postprocessing algorithm was developed. It corrects for wave front aberration-induced image plane deformation and for background and noise signals, enabling us to observe subcellular processes. Exemplarily, we quantitatively and qualitatively analyze angiogenesis in bone regeneration. We make use of a transgenic reporter mouse strain with nucleargreen fluorescent protein and membrane-bound tdTomato under the Cadherin-5 promoter. We observe two phases of vascularization. First, rapid vessel sprouting pervades the FOV within 3-4 days after osteotomy. Second, the vessel network continues to be dynamically remodeled until the end of our observation time, 14 days after surgery. Limbostomy opens a unique set of opportunities and allows further insight on spatiotemporal aspects of bone marrow biology, for example, hematopoiesis, analysis of cellular niches, immunological memory, and vascularization in the bone marrow during health and disease. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Public transport plays a vital role in society as the economy, the degree of sustainability and the qualityof life of a city is directly affected by transportation. A shift in modal share towards public transport isassociated with many benefits such as increased air quality and improved space allocation within thecity. To further promote public transport, an appropriate measure of competitiveness is required toevaluate the impact of past and future transport policies. This study introduces the journeys per capitaratio as a new way of measuring public transport competitiveness. Firstly, the key factors affecting thepublic transportation usage rate expressed as the journeys per capita ratio are identified to evaluatethe impact of public transport provider efforts. For this purpose, data for a total of 32 explanatoryvariables and a scope of 218 regions for seven consecutive time frames are collected. Secondly,geographically weighted regression (GWR) – a local regression-based spatial analysis technique – isperformed to test if the journeys per capita ratio is a suitable target variable to predict the impact ofcertain transport supply changes. A traditional global ordinary least square (OLS) model is conductedas well to compare if a local model could be more beneficial. The GWR and the OLS model are trainedwith the data of previous years and tested with data from the consecutive following years. Thirdly,further temporal and socio-economic based cluster analyses are performed to assess the validity andthe explanatory power of the journeys per capita ratio. The conducted analyses reveal that thejourneys per capita ratio is a superior measure for assessing public transport competitiveness.Goodness of fit statistics and estimation results demonstrate that the GWR model has betterprediction accuracy and is more capable of retrospectively predicting the impact of previous transportpolicies. Kollektivtrafiken har en avgörande roll i samhället då ekonomin, graden av hållbarhet och städerslivskvalité är direkt påverkad av transport. En förändring av transportanvändning från bil motkollektivtrafik är förknippad med flera fördelar, såsom ökad luftkvalitet och förbättrad rumsligallokering inom staden. För att ytterligare främja kollektivtrafik krävs ett lämpligt mått påkonkurrenskraft för att utvärdera effekterna av tidigare och framtida transportpolitik. Den här studienintroducerar resor per capita-förhållanden som ett nytt sätt att mäta kollektivtransportenskonkurrenskraft. För det första identifieras nyckelfaktorerna som påverkar användningsgraden förkollektivtrafik, uttryckt som förhållandet resor per capita för att utvärdera effekten avkollektivtrafikleverantörens insatser. För det här syftet har data för totalt 32 variabler och ett omfångav 218 regioner under sju, på varandra, följande tidsramar har samlats in. För det andra har Geografisktviktad regression (GWR), vilket är en lokal regressionsbaserad rumslig analysteknik, använts för atttesta om resor per capita-förhållanden är en lämplig målvariabel för att förutsäga effekterna av vissatransportförändringar. En traditionell Global ordinary least square model (OLS) har också använts föratt jämföra om en lokal modell är mer fördelaktig. GWR och OLS modellerna har tränats med data fråntidigare år och testats med data från följande år. För det tredje har ytterligare tidsmässigsocioekonomisk baserad klusteranalys utförts för att bedöma validiteten och förklaringsförmågan förresornas förhållande per capita. De genomförda analyserna pekar på att förhållandet resor per capitaär ett fördelaktigt mått för att bedöma kollektivtrafikens konkurrenskraft. Goodness of fit statistics ochde uppskattade resultaten visar att GWR-modellen har en bättre förmåga att göra noggrannaförutsägelser och är mer kapabel att i efterhand förutsäga effekterna av tidigare transportpolitik.

Two-photon microscopy (2PM) has brought unique insight into the mechanisms underlying immune system dynamics and function since it enables monitoring of cellular motility and communication in complex systems within their genuine environment-the living organism. However, use of 2PM in clinical settings is limited. In contrast, optical coherence tomography (OCT), a noninvasive label-free diagnostic imaging method, which allows monitoring morphologic changes of large tissue regions in vivo, has found broad application in the clinic. Here we developed a combined multimodal technology to achieve near-instantaneous coregistered OCT, 2PM, and second harmonic generation (SHG) imaging over large volumes (up to 1,000 × 1,000 × 300 μm3 ) of tendons and other tissue compartments in mouse paws, as well as in mouse lymph nodes, spleens, and femurs. Using our multimodal imaging approach, we found differences in macrophage cell shape and motility behavior depending on whether they are located in tendons or in the surrounding tissue compartments of the mouse paw. The cellular shape of tissue-resident macrophages, indicative for their role in tissue, correlated with the supramolecular organization of collagen as revealed by SHG and OCT. Hence, the here-presented approach of coregistered OCT and 2PM has the potential to link specific cellular phenotypes and functions (as revealed by 2PM) to tissue morphology (as highlighted by OCT) and thus, to build a bridge between basic research knowledge and clinical observations. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Scripta materialia 127, 151 - 155(2017). doi:10.1016/j.scriptamat.2016.09.026, We report a synchrotron in situ diffraction experiment exploring stress evolution during compression of interpenetrating-phase nanocomposites based on nanoporous gold and polymer.While previous experiments providedindirect indication of local flow conditions based on macroscopic effective flow stress and micromechanicsmodels such as Gibson-Ashby scaling law, the lattice parameter data of our experiment access the flow stress directly.At small structure size we find excellent agreement with previous reports, supporting thematch betweenmaterial andmodel of those studies. Yet, deviations at larger structure size suggest that coarsening generates defectsin metal network structure that are ignored by standard models., Published by Elsevier Science, Amsterdam [u.a.]

Protoneutron stars (PNS) formed in core-collapse supernovae (CCSNe) may contain an abundant number of muons due to the high temperatures and the high electron chemical potentials. The appearance of muons “softens” the PNS equation of state, triggers faster PNS contraction, and thus leads to increased luminosities and mean energies of all emitted neutrinos. In this thesis, I present the first self-consistent implementation of muons into CCSN simulations and their effect on the delayed neutrino-driven explosion mechanism. Die hohen Temperaturen und chemischen Potentiale der Elektronen in den bei Kernkollaps-Supernovae (KKSN) auftretenden Protoneutronen Sternen erlauben eine große Zahl von Myonen im dichten Medium. Ihr Auftreten macht die Zustandsgleichung “weicher”, was zur schnelleren Kontraktion und damit zu erhöhten Luminositäten und mittleren Energien aller emittierten Neutrinos führt. In dieser Dissertation bespreche ich die erste selbstkonsistente Implementierung von Myonen in KKSN Simulationen und ihre Auswirkungen auf den neutrino-getriebenen Explosionsmechanismus.

The bone marrow is a central organ of the immune system, which hosts complex interactions of bone and immune compartments critical for hematopoiesis, immunological memory, and bone regeneration. Although these processes take place over months, most existing imaging techniques allow us to follow snapshots of only a few hours, at subcellular resolution. Here, we develop a microendoscopic multi-photon imaging approach called LIMB (longitudinal intravital imaging of the bone marrow) to analyze cellular dynamics within the deep marrow. The approach consists of a biocompatible plate surgically fixated to the mouse femur containing a gradient refractive index lens. This microendoscope allows highly resolved imaging, repeatedly at the same regions within marrow tissue, over months. LIMB reveals extensive vascular plasticity during bone healing and steady-state homeostasis. To our knowledge, this vascular plasticity is unique among mammalian tissues, and we expect this insight will decisively change our understanding of essential phenomena occurring within the bone marrow., Longitudinal imaging of bone marrow would shed insight into long-term cellular dynamics within this compartment. Here, the authors develop a multi-photon imaging approach for the mouse femur and reveal extensive vascular plasticity within the bone marrow during bone healing and steady-state homeostasis.

A new compound class of diaryl-substituted heterocycles with tricyclic dihydropyrrolo[3,2,1-hi]indole and pyrrolo[3,2,1-hi]indole core structures has been designed and was synthesized by a modular sequence of Friedel-Crafts acylation, amide formation, and McMurry cyclization. This synthesis route represents a novel and versatile access toward dihydropyrrolo[3,2,1-hi]indoles and is characterized by good chemical yields and high modularity. From a set of 19 derivatives, 11 candidates were selected for determination of their COX inhibition potency and were found to be selective inhibitors with high affinity to COX-2 (IC50 ranging from 20-2500 nM and negligible inhibition of COX-1). The binding mode of the novel inhibitors in the active side of COX-2 was calculated in silico using the protein-ligand docking program GOLD by application of the molecular structures of two compounds derived from X-ray crystallography. Two novel compounds with high affinity to COX-2 (6k = 70 nM, 8e = 60 nM) have a fluoro substituent, making them promising candidates for the development of (18)F-radiolabeled COX-2 inhibitors for imaging purposes with positron emission tomography (PET).

When rendering complex scenes using path-tracing methods, long processing times are required to calculate a sufficient number of samples for high quality results. In this paper, we propose a new method for priority sampling in path-tracing that exploits restrictions of the human visual system by recognizing whether an error is perceivable or not. We use the stationary wavelet transformation to efficiently calculate noise-contrasts in the image based on the standard error of the mean. We then use the Contrast Sensitivity Function and Contrast Masking of the Human Visual System to detect if an error is perceivable for any given pixel in the output image. Errors that can not be detected by a human observer are then ignored in further sampling steps, reducing the amount of samples calculated while producing the same perceived quality. This approach leads to a drastic reduction in the total number of samples required and therefore in total rendering time.

In aging individuals, both protective as well as regulatory immune functions are declining, resulting in an increased susceptibility to infections as well as to autoimmunity. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2-deficiency in immune cell subsets has been shown to be associated with aging. Using intravital marker-free NAD(P)H-fluorescence lifetime imaging, we have previously identified microglia/myeloid cells and astrocytes as main cellular sources of NADPH oxidase (NOX) activity in the CNS during neuroinflammation, due to an overactivation of NOX. The overactivated NOX enzymes catalyze the massive production of the highly reactive O−2, which initiates in a chain reaction the overproduction of diverse reactive oxygen species (ROS). Age-dependent oxidative distress levels in the brain and their cellular sources are not known. Furthermore, it is unclear whether in age- dependent diseases oxidative distress is initiated by overproduction of ROS or by a decrease in antioxidant capacity, subsequently leading to neurodegeneration in the CNS. Here, we compare the activation level of NOX enzymes in the cerebral cortex of young and aged mice as well as in a model of vascular amyloid pathology. Despite the fact that a striking change in the morphology of microglia can be detected between young and aged individuals, we find comparable low-level NOX activation both in young and old mice. In contrast, aged mice with the human APPE693Q mutation, a model for cerebral amyloid angiopathy (CAA), displayed increased focal NOX overactivation in the brain cortex, especially in tissue areas around the vessels. Despite activated morphology in microglia, NOX overactivation was detected only in a small fraction of these cells, in contrast to other pathologies with overt inflammation as experimental autoimmune encephalomyelitis (EAE) or glioblastoma. Similar to these pathologies, the astrocytes majorly contribute to the NOX overactivation in the brain cortex during CAA. Together, these findings emphasize the role of other cellular sources of activated NOX than phagocytes not only during EAE but also in models of amyloid pathology. Moreover, they may strengthen the hypothesis that microglia/monocytes show a diminished potential for clearance of amyloid beta protein.

Simultaneous detection of multiple cellular and molecular players in their native environment, one of the keys to a full understanding of immune processes, remains challenging for in vivo microscopy. Here, we present a synergistic strategy for spectrally multiplexed in vivo imaging composed of (i) triple two-photon excitation using spatiotemporal synchronization of two femtosecond lasers, (ii) a broad set of fluorophores with emission ranging from blue to near infrared, (iii) an effective spectral unmixing algorithm. Using our approach, we simultaneously excite and detect seven fluorophores expressed in distinct cellular and tissue compartments, plus second harmonics generation from collagen fibers in lymph nodes. This enables us to visualize the dynamic interplay of all the central cellular players during germinal center reactions. While current in vivo imaging typically enables recording the dynamics of 4 tissue components at a time, our strategy allows a more comprehensive analysis of cellular dynamics involving 8 single-labeled compartments. It enables to investigate the orchestration of multiple cellular subsets determining tissue function, thus, opening the way for a mechanistic understanding of complex pathophysiologic processes in vivo. In the future, the design of transgenic mice combining a larger spectrum of fluorescent proteins will reveal the full potential of our method.

A critical drawback for the application of magnesium wrought alloys is the limited formability of semifinished products that arises from a strong texture formation during thermomechanical treatment. The ability of second phase particles embedded into the metal matrix to alter this texture evolution is of great interest. Therefore, the fabrication of particle modified magnesium alloys (particle content 0.5–1 wt.-%) by gravity die casting has been studied. Five different types of micron sized ceramic powders (AlN, MgB2, MgO, SiC, and ZrB2) have been investigated to identify applicable particles for the modification. Agglomeration of the particles is revealed to be the central problem for the fabrication process. The main factors that influence the agglomerate size are the particle size and the intensity of melt stirring. Concerning handling, chemical stability in the Mg-Al-Zn alloy system, settling and wetting in the melt, and formation of the microstructure in most cases, the investigated powders show satisfying properties. However, SiC is chemically unstable in aluminum containing alloys. The high density of ZrB2 causes large particles to settle subsequent to stirring resulting in an inhomogeneous distribution of the particles over the cast billet.

A hallmark of autoimmune retinal inflammation is the infiltration of the retina with cells of the innate and adaptive immune system, leading to detachment of the retinal layers and even to complete loss of the retinal photoreceptor layer. As the only optical system in the organism, the eye enables non-invasive longitudinal imaging studies of these local autoimmune processes and of their effects on the target tissue. Moreover, as a window to the central nervous system (CNS), the eye also reflects general neuroinflammatory processes taking place at various sites within the CNS. Histological studies in murine neuroinflammatory models, such as experimental autoimmune uveoretinitis (EAU) and experimental autoimmune encephalomyelitis, indicate that immune infiltration is initialized by effector CD4(+) T cells, with the innate compartment (neutrophils, macrophages, and monocytes) contributing crucially to tissue degeneration that occurs at later phases of the disease. However, how the immune attack is orchestrated by various immune cell subsets in the retina and how the latter interact with the target tissue under in vivo conditions is still poorly understood. Our study addresses this gap with a novel approach for intravital two-photon microscopy, which enabled us to repeatedly track CD4(+) T cells and LysM phagocytes during the entire course of EAU and to identify a specific radial infiltration pattern of these cells within the inflamed retina, starting from the optic nerve head. In contrast, highly motile [Formula: see text] cells display an opposite radial motility pattern, toward the optic nerve head. These inflammatory processes induce modifications of the microglial network toward an activated morphology, especially around the optic nerve head and main retinal blood vessels, but do not affect the neurons within the ganglion cell layer. Thanks to the new technology, non-invasive correlation of clinical scores of CNS-related pathologies with immune infiltrate behavior and subsequent tissue dysfunction is now possible. Hence, the new approach paves the way for deeper insights into the pathology of neuroinflammatory processes on a cellular basis, over the entire disease course.

With the aim of imaging and quantification of oxytocin receptors (OTRs) in living brain using positron emission tomography (PET), we developed a (18)F-labeled small molecule radiotracer and investigated its in vivo pharmacokinetics in mice and pig. [(18)F]6b (KD = 12.3 nM) was radiolabeled by a two-step procedure using a microwave system with radiochemical yields of 26.9 ± 4.7%. Both organ distribution and small animal PET studies revealed limited brain uptake of [(18)F]6b in mouse (mean SUV of 0.04 at 30 min pi). Besides, significant radioactivity uptake in the pituitary gland was observed (SUV of 0.7 at 30 min pi). In a dynamic PET study in one piglet, we detected a higher uptake of [(18)F]6b in the olfactory bulb (SUV of 0.34 at 30 min pi) accompanied by a low uptake in the whole brain. In vitro autoradiographic studies on porcine brain sections indicated interaction of [(18)F]6b with several off-target receptors.

Radiopharmacological investigations of [(18)F]NS14490 have proven that this radiotracer could be a potential PET radiotracer for imaging of alpha7 nicotinic acetylcholine receptor particularly with regard to vulnerable plaques of diseased vessels. For further optimisation of the previously automated one-pot radiosynthesis of [(18)F]NS14490 using a tosylate precursor, precursors with other leaving groups (nosylate and mosylate) were synthesized and compared with the tosylate with respect to their reactivities towards [(18)F]fluoride. The use of these different precursors resulted in comparable labelling yields of [(18)F]NS14490. A novel mosylate precursor was synthesized and evaluated, which has revealed a higher stability during a storage period of five months compared to the corresponding tosylate and nosylate.

The development of intravital Förster Resonance Energy Transfer (FRET) is required to probe cellular and tissue function in the natural context: the living organism. Only in this way can biomedicine truly comprehend pathogenesis and develop effective therapeutic strategies. Here we demonstrate and discuss the advantages and pitfalls of two strategies to quantify FRET in vivo—ratiometrically and time-resolved by fluorescence lifetime imaging—and show their concrete application in the context of neuroinflammation in adult mice.