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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.

Infections with intestinal nematodes have an ambivalent impact: they represent a burden for human health and animal husbandry, but, at the same time, may ameliorate auto-immune diseases due to the immunomodulatory effect of the parasites. Thus, it is key to understand how intestinal nematodes arrive and persist in their luminal niche and interact with the host over long periods of time. The basic mechanism ruling over parasite and host cellular and tissue functions, the metabolism, was largely neglected in the study of intestinal nematode infections. Here we use NADH (nicotinamide adenine dinucleotide) and NADPH (nicotinamide adenine dinucleotide phosphate) fluorescence lifetime imaging of explanted murine duodenum infected with the natural nematode Heligmosomoides polygyrus and define the link between general metabolic and specific enzymatic activity in parasite and host tissue, during acute infection. In both healthy and infected host intestine, energy is effectively produced, mainly via oxidative phosphorylation. In contrast, the nematodes shift their energy production from balanced fast anaerobic glycolysis and effective oxidative phosphorylation, towards mainly anaerobic glycolysis, back to oxidative phosphorylation during the different life cycle phases in the submucosa versus the intestinal lumen. Additionally, we found an increased NADPH oxidase (NOX) enzymes-dependent oxidative burst in infected intestinal host tissue as compared to healthy tissue, which was mirrored by a similar defense reaction in the parasites. We expect that, the here presented application of in vivo NAD(P)H-FLIM constitutes a unique tool to study metabolic host-parasite crosstalk, in various parasitic intestinal infections.

Infections with intestinal nematodes have an equivocal impact: they represent a burden for human health and animal husbandry, but, at the same time, may ameliorate auto-immune diseases due to the immunomodulatory effect of the parasites. Thus, it is key to understand how intestinal nematodes arrive and persist in their luminal niche and interact with the host over long periods of time. One basic mechanism governing parasite and host cellular and tissue functions, metabolism, has largely been neglected in the study of intestinal nematode infections. Here we use NADH (nicotinamide adenine dinucleotide) and NADPH (nicotinamide adenine dinucleotide phosphate) fluorescence lifetime imaging of explanted murine duodenum infected with the natural nematode Heligmosomoides polygyrus and define the link between general metabolic activity and possible metabolic pathways in parasite and host tissue, during acute infection. In both healthy and infected host intestine, energy is effectively produced, mainly via metabolic pathways resembling oxidative phosphorylation/aerobic glycolysis features. In contrast, the nematodes shift their energy production from balanced fast anaerobic glycolysis-like and effective oxidative phosphorylation-like metabolic pathways, towards mainly anaerobic glycolysis-like pathways, back to oxidative phosphorylation/aerobic glycolysis-like pathways during their different life cycle phases in the submucosa versus the intestinal lumen. Additionally, we found an increased NADPH oxidase (NOX) enzymes-dependent oxidative burst in infected intestinal host tissue as compared to healthy tissue, which was mirrored by a similar defense reaction in the parasites. We expect that, the here presented application of NAD(P)H-FLIM in live tissues constitutes a unique tool to study possible shifts between metabolic pathways in host-parasite crosstalk, in various parasitic intestinal infections.

The use of modern medical equipment in crisis and war zones for emergency medical teams (EMT) of the World Health Organization is an important factor for fast and efficient humanitarian aid. A reliable vital parameter monitoring is fundamental in mobile hospitals. Currently, the maintenance of medical devices in structurally weak areas is difficult due to the company’s proprietary standards. Rough environmental influences such as dust, moisture, heat or shocks can lead to dysfunktion and long-lasting failure of instrumentation. Pulse oximetry and blood pressure measurements are particularly susceptible. We developed an open source vital parameter monitoring system for use under adverse conditions and structurally weak areas. Blood oxygen levels, heart rate, blood pressure and electrocardiograms are recorded and transferred to decentralized displays. The main focus is on reliability and robustness of various optical sensors for pulse oximetry, the repair capability of the system also for non-technical personnel and the availability of individual standard components. Therefore we implemented a monitoring system basing on individual microcontrollers for each vital parameter. Different optical sensors for measurement in transmission and reflection were tested at suitable body sites with near-surface arteries. In combination with the electrocardiogram, evaluation of the pulse transit time enables continuous blood pressure measurements. A specially developed optical reflective sensor allows reliable measurement of blood oxygen level. For extended blood pressure measurements, the pulsetransit-time method (PTT) was implemented and enables a continuous monitoring. Even in emergencies, the trend in blood pressure can be monitored with PTT without prior calibration. The reliability was investigated.