Project Details
Projekt
Intravital nanoscopy for investigating the blood-brain barrier
Subject Area
Pharmacology
Term
from 2019 to 2023
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 413932741
The mammalian brain is a highly plastic organ that adapts to changes in the environment. Functional plasticity in the nervous system is often tightly coupled to structural changes that can be visualized in living organisms by optical microscopy. Although this technology has had a major impact on neuroscience, many important structural changes occur at the subcellular and molecular level and are smaller than 250 nm, the resolution limit of optical microscopy. However, recent ground-breaking innovations exploit the photophysical properties of fluorophores and extend the scope of light microscopy into the nanometer range. These new technologies are summarized under the term optical nanoscopy. Among the various nanoscopy approaches, stimulated emission depletion (STED1) nanoscopy is compatible with intravital imaging. While several groups have used STED nanoscopy for imaging subcellular processes in cultured cells, a few pioneering studies have applied this technique in vivo providing proof of principle that STED nanoscopy is feasible for investigating the living brain. In most cases, however, further optimization and standardization of the technology are required for exploiting its full biological potential. Building on our expertise in intravital imaging we will contribute to this task by pursuing three specific technical aims: we plan to facilitate the retrieval of target sites in STED imaging; we want to correct for motion and aberration artefacts and we seek to reduce photodamage. To accomplish these aims we will integrate optic coherence tomography (OCT) and multiphoton techniques into the STED setup. In parallel, we will put intravital STED nanoscopy to the test and apply it to the investigation of the blood-brain barrier (BBB) at the subcellular level. In this clinically important field intravital STED nanoscopy has not been used so far. We are convinced that the technology is perfectly suitable to solve some key problems that have hampered previous BBB research. Importantly, the polarity of the barrier occurs at a dimension that is beyond the resolution of conventional light microscopy. At the same time, the barrier is highly dynamic and plastic, a feature that has confounded static high resolution imaging with electron microscopy. Cerebral capillaries in the superficial layers of the cortex are accessible for intravital STED nanoscopy. Exploiting these strong points we plan to investigate the polarity of transporters in the BBB, transcytosis across the BBB, and the interaction of endothelial cells with pericytes. In addition, cooperating with groups in Bern and Hamburg we will investigate the transmigration of leukocytes across the BBB and the neuronal and synaptic consequences of BBB disruption. The nanoscope will be set up within the Small Animal Imaging Lübeck (SAIL) facility, making the instrumentation and the know-how gained with intravital STED imaging available to other researches inside and outside of Lübeck.
DFG Programme
Major Instrumentation Initiatives
Major Instrumentation
Intravital-2-Photon-STED-Microscope
Instrumentation Group
5090 Spezialmikroskope
Applicant Institution
Universität zu Lübeck
Participating Persons
Professor Dr. Gereon Hüttmann; Professor Dr. Peter König
