multiphoton-intravital-imaging-for-monitoring-leukocyte-recruitment

Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for ...

University of Bern

The development of multi-photon intravital microscopy, in particular two-photon microscopy (2PM), has been a breakthrough technique for deep-tissue imaging of dynamic cell behavior inside live organisms and has substantially advanced the field of immunology. However, intravital time-lapse imaging over prolonged time periods is complicated by slow tissue drifts caused by vital activity, leading to shifting fields of views and making the acquired image sequence partially or completely unanalyzable. To solve this issue, we have established a system that performs continuous drift offset correction in real time using fine pattern matching during 2PM acquisition. We incorporated an extensive use of graphical processing unit (GPU) for high-speed computing required for real time correction during data acquisition. This allowed us to perform prolonged acquisitions and increase the proportion of analyzable datasets to nearly 100% in lymphoid and non-lymphoid tissues. Considering the straightforward implementation of our newly developed system, we anticipate that it will be applicable for other users interested in improving the quality of live imaging data acquisition.

Real-time tissue offset correction system for intravital multiphoton microscopy.

T cells are actively scanning pMHC-presenting cells in lymphoid organs and nonlymphoid tissues (NLTs) with divergent topologies and confinement. How the T cell actomyosin cytoskeleton facilitates this task in distinct environments is incompletely understood. Here, we show that lack of Myosin IXb (Myo9b), a negative regulator of the small GTPase Rho, led to increased Rho-GTP levels and cell surface stiffness in primary T cells. Nonetheless, intravital imaging revealed robust motility of Myo9b CD8 T cells in lymphoid tissue and similar expansion and differentiation during immune responses. In contrast, accumulation of Myo9b CD8 T cells in NLTs was strongly impaired. Specifically, Myo9b was required for T cell crossing of basement membranes, such as those which are present between dermis and epidermis. As consequence, Myo9b CD8 T cells showed impaired control of skin infections. In sum, we show that Myo9b is critical for the CD8 T cell adaptation from lymphoid to NLT surveillance and the establishment of protective tissue-resident T cell populations.

The Rho regulator Myosin IXb enables nonlymphoid tissue seeding of protective CD8 T cells.

The homeostasis of the central nervous system (CNS) is ensured by the endothelial, epithelial, mesothelial and glial brain barriers, which strictly control the passage of molecules, solutes and immune cells. While the endothelial blood-brain barrier (BBB) and the epithelial blood-cerebrospinal fluid barrier (BCSFB) have been extensively investigated, less is known about the epithelial and mesothelial arachnoid barrier and the glia limitans. Here, we summarize current knowledge of the cellular composition of the brain barriers with a specific focus on describing the molecular constituents of their junctional complexes. We propose that the brain barriers maintain CNS immune privilege by dividing the CNS into compartments that differ with regard to their role in immune surveillance of the CNS. We close by providing a brief overview on experimental tools allowing for reliable in vivo visualization of the brain barriers and their junctional complexes and thus the respective CNS compartments.

Article	Year
Real-time tissue offset correction system for intravital multiphoton microscopy. Journal of immunological methods\| PubMed ID: 27589923	2016
The Rho regulator Myosin IXb enables nonlymphoid tissue seeding of protective CD8 T cells. The Journal of experimental medicine\| PubMed ID: 29875261	2018
Structure and Junctional Complexes of Endothelial, Epithelial and Glial Brain Barriers. International journal of molecular sciences\| PubMed ID: 31671721	2019

Mykhailo Vladymyrov

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