Studying interaction of the immune and vascular system using intravital multiphoton microscopy

With the beginning of the new millennium intravital multiphoton imaging of the immune system has become a necessary tool for understanding cellular behavior in steady state and models of disease, in vivo . The first studies visualized dynamics of antigen presenting cells and T‐cells. Followed by development of fluorescent reporter mice that allowed tracking of neutrophils, monocytes and macrophages, however the spatiotemporal resolution achieved wasn't sufficient enough to study physiological parameters like blood or lymph flow. We have improved our two‐photon imaging approach by decreasing photo toxicity and generated a combination of fluorophores which are low in cytotoxicity, able to label morphological structures and cell populations of interest. This combination of dyes is based on high excitation efficiency capable to be excited with low laser power and bright emission properties. Together with our customized dual femtosecond pulsed laser system (InSight DS+ together with an Mai Tai DeepSee) featuring an ultra‐fast resonance scanner and sensitive hybrid detectors, we can image up to five fluorophores in intravital settings. This approach enables us to visualize lymphatics and blood vessels together with myeloid cells and lymphocytes in vivo examining changes in flow dynamics and cellular interaction (Fig 1). For decades blood vasculature has been studied extensively, but our understanding of lymphatic functions remains poor. Lymphatics interact systemically and locally with the blood vasculature (Fig 2) and using these technique we have recently shown that dendritic cells are an important regulator in lymphatic function (Ivanov et. al. J Clin Invest. 2016 Apr 1;126(4):1581–91.). Many immune‐vasculature interactions can only be proven in the living animal, showing the interactions and impact of immune cells on vascular flow dynamics and vice versa. Our most recent intravital imaging studies allow the visualization of arterial resident macrophage dynamics within atherosclerotic plaque. Additionally, we are utilizing our intravital vascular imaging approaches to address the challenge of determining the role of distinct subsets of myeloid cells in glomerular disease and in murine tumor models (Fig 3). Furthermore, we are hoping these studies help to identify functional interactions of the vasculature and different immune cell populations as therapeutically targets for inflammatory diseases and for the enhancement of cancer immune therapy. Support or Funding Information BHZ was awarded a Scientist Development Grant (16SDG30480008) from the American Heart Association (AHA) 07/01/16‐06/30/19