Application to the Analysis of Germinal Center Reactions In Vivo

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

Limbostomy: Longitudinal Intravital Microendoscopy in Murine Osteotomies

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

Longitudinal intravital imaging of the femoral bone marrow reveals plasticity within marrow vasculature.

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

Longitudinale intravitale Multiphotonen-Mikroendoskopie im Knochenmark von Mäusen

Bone forming cells of mesenchymal origin and hematopoietic cells of the immune system interact in the bone marrow. Both cell types show high plasticity under homeostatic conditions as well as during bone healing after an injury. Bone marrow is also the central organ for hematopoiesis and harbors the immunological memory. Above all, regeneration and formation of an immunological memory are processes that take place over a period of weeks to months. Since the underlying mechanisms are controlled on the cellular level, in vivo multiphoton fluorescence microscopy is the most appropriate and common method to gain insight into the dynamics of these interactions in living tissue under physiological and pathological conditions. However, already established methods for intravital microscopy in the bone marrow of mice are, either designed for imaging a few hours and thus not for repeated, i.e. longitudinal observations, or not designed for long bones, but only for flat bones, i.e. calvarium. In addition, they are physically limited to penetration depths of approx. 100 - 150 µm below the bone cortex. In the present work, in order to fulfil the needs in understanding bone biology a new microendoscopic technique based on gradient index (GRIN) lenses was developed and applied to analyze cellular dynamics in vivo. An internal fixation system ensured the precise positioning of a GRIN endoscope in the femoral bone marrow tissue over months. The optical performance of the endoscope system was comparable to other methods for intravital microscopy in the bone marrow. Via a chronic window, which can be introduced into the bone marrow cavity both centrally and pericortically, we were able for the first time to observe and quantify changes in the vascular structure in one and the same individual over month. The observed plasticity of the blood vessels seems to be unique compared to other tissues. Using histological and flow cytometric methods, we could exclude the possibility that this is a process driven by regeneration processes caused by implantation procedure. Additionally, to account for the complex dynamic interactions between various cellular and extra-cellular compartments, a method for non-linear asymmetric two-photon excitation (ATPE) and digital unmixing of the recorded fluorescence signals was established, which can be used for the multiplexed recording of eight different tissue components. The novel endoscope system enables longitudinal intravital microscopy in the murine bone marrow (LIMB) to analyze the osteoimmune system in vivo under homeostatic and pathological conditions. With LIMB we expect to gain a deeper insight into the role of the immune system in bone healing, as well as into the establishment and maintenance of immunological memory cells, such as long-lived plasma cells in their special survival niches.Knochenbildende Zellen mesenchymalen Ursprungs und hämatopoetische Zellen des Immunsystems interagieren im Knochenmark. Sie besitzen sowohl unter homöostatischen Bedingungen als auch während der Knochenheilung nach einer Fraktur eine hohe Plastizität. Das Knochenmark ist das zentrale Organ für die Hämatopoese und unser immunologisches Gedächtnis. Knochenregeneration und die Bildung des immunologischen Gedächtnisses sind Vorgänge, die innerhalb von Wochen oder Monaten ablaufen. Da die zugrundeliegenden Mechanismen auf zellulärer Ebene kontrolliert werden, ist die in vivo Multiphotonen-Mikroskopie die geeignetste Methode, um einen Einblick in die dynamischen Interaktionen im lebenden Gewebe unter physiologischen und pathologischen Bedingungen zu gewinnen. Die bereits etablierten Methoden zur Intravitalmikroskopie im Knochenmark von Mäusen sind jedoch zum einen entweder auf Aufnahmen über wenige Stunden und nicht für wiederholte, d.h. longitudinale Beobachtungen ausgelegt, sowie physikalisch auf Eindringtiefen von ca. 100 - 150 µm beschränkt, oder lassen sich nicht auf Röhrenknochen anwenden. Um den Anforderungen der Analyse der Knochenbiologie entgegenzutreten, wurde eine neue mikro-endoskopische Technik, die auf Gradienten-Index-Linsen (GRIN-Linsen) basiert, entwickelt und getestet. Dabei wurde ein interner Fixateur verwendet, welcher über Monate die präzise Positionierung eines GRIN-Endoskops im Knochenmarksgewebe des Femurs von Mäusen gewährleistet. Die optische Leistungsfähigkeit des Endoskops war mit anderen Methoden zur Intravitalmikroskopie im Knochenmark vergleichbar. Mit Hilfe des Endoskops, welches sowohl zentral als auch perikortikal ins Knochenmark eingebracht werden kann, waren wir erstmals in der Lage Veränderungen in der Gefäßstruktur in ein und demselben Individuum über Monate zu verfolgen und zu quantifizieren. Es zeigte sich eine hohe Plastizität der Blutgefäße, welche im Vergleich zu anderen Geweben einzigartig zu sein scheint. Wir konnten dabei mittels histologischer und durchflusszytometrischer Methoden ausschließen, dass es sich um einen durch Regenerationsprozesse getriebenen Vorgang handelt, der als Reaktion auf die Implantation des Systems ausgelöst wurde. Um die komplexen, dynamischen Wechselwirkungen zwischen verschiedenen zellulären und extrazellulären Kompartimenten in der intravitalen Bildgebung zu berücksichtigen, wurde des Weiteren ein Verfahren zur nichtlinearen asymmetrischen Zwei-Photonen-Anregung (ATPE) und der digitalen Entfaltung der aufgenommenen Fluoreszenzsignale etabliert, welches zur parallelen Aufnahme von acht verschiedenen Gewebekompartimenten genutzt werden kann. Damit ermöglicht das neuartige Endoskopsystem die longitudinale Intravitalmikroskopie im murinen Knochenmark (LIMB) zur Analyse des Osteo-Immunsystems in vivo unter homöostatischen und pathologischen Bedingungen. Mit LIMB erhoffen wir uns einen tieferen Einblick in die Rolle des Immunsystems im Verlauf der Knochenheilung, sowie in die Etablierung und des Erhalts immunologischer Gedächtniszellen, wie z.B. langlebiger Plasmazellen in den speziellen Überlebensnischen, zu gewinnen