New advancements in highly sensitive time-resolved fluorescence two-photon microscopy: Theoretical approaches and bioscientific applications

A central methodological aim of the biosciences is to experimentally simulate, as good as possible, the real environmental conditions of biological systems in order to gain a true image of the effects and processes of interest. Thus, the bioscientific research is moving from test-tube observations towards in vivo investigations in intact cells and tissues. In this frame, the development of biophysical techniques, which allow an accurate and highly sensitive registration of changes in the studied system without disturbing the natural processes therein, is particularly significant. Especially the fluorescence laser microscopy provide a large diversity of versatile tools for bioscientific studies. In this work, two new theoretical approaches are developed, which allow an extended comprehension of the single-molecule fluorescence fluctuation microscopy based on two-photon excitation, one of the most sensitive techniques for biological investigations. Moreover, novel non-invasive fluorescence lifetime imaging experiments are performed, which enable us a detailed and accurate insight in the cellular NAD(P)H-metabolism and in the functionality of artificial skin as an organ.Eines der Hauptziele in den Biowissenschaften ist es, die tatsächliche Umgebung biologischer Systeme experimentell zu simulieren, um eine naturgetreue Abbildung der wichtigen Effekte und Prozesse in diesen Systemen zu erhalten. Aus diesem Grund entfernt sich die biowissenschaftliche Forschung von Untersuchungen im Reagenzglas und widmet zunehmend den Experimenten in intakten Zellen und Geweben Aufmerksamkeit. In diesem Zusammenhang ist die Entwicklung biophysikalischer Verfahren besonders wichtig, die eine sehr genaue und hoch-sensitive Aufnahme der Veränderungen in dem zu untersuchenden System erlauben, ohne dass die darin ablaufenden natürlichen Prozesse gestört werden. Besonders die Fluoreszenz-Laser-Mikroskopie stellt eine große Auswahl an Verfahren für biowissenschaftliche Untersuchungen zu Verfügung. In dieser Arbeit werden zwei neue theoretische Modelle vorgestellt, die ein detailliertes Verständnis der, auf Zwei-Photonen-Anregung basierenden, Einzelmolekül-Nachweis-Fluoreszenz-Fluktuations-Mikroskopie ermöglichen. Diese Methode stellt eine der empfindlichsten und genausten Methoden für biologische Anwendungen dar. Außerdem werden neuartige, gewebeschonende Fluoreszenz-Lebensdauer-Imaging Experimente vorgestellt, die eine umfangreiche und quantitative Einsicht in den NAD(P)H Zellmetabolismus und in die Funktionalität künstlicher Haut als Organ zulassen

Analyzing nicotinamide adenine dinucleotide phosphate oxidase activation in aging and vascular amyloid pathology

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

MarShie: a clearing protocol for 3D analysis of single cells throughout the bone marrow at subcellular resolution

Abstract Analyzing immune cell interactions in the bone marrow is vital for understanding hematopoiesis and bone homeostasis. Three-dimensional analysis of the complete, intact bone marrow within the cortex of whole long bones remains a challenge, especially at subcellular resolution. We present a method that stabilizes the marrow and provides subcellular resolution of fluorescent signals throughout the murine femur, enabling identification and spatial characterization of hematopoietic and stromal cell subsets. By combining a pre-processing algorithm for stripe artifact removal with a machine-learning approach, we demonstrate reliable cell segmentation down to the deepest bone marrow regions. This reveals age-related changes in the marrow. It highlights the interaction between CX3CR1+ cells and the vascular system in homeostasis, in contrast to other myeloid cell types, and reveals their spatial characteristics after injury. The broad applicability of this method will contribute to a better understanding of bone marrow biology