Increasing the palette of fluorophores for intravital 2P-imaging of the vascular and immune system in mice

Abstract

Abstract Disruption in the movement of blood or lymph leads to potentially fatal diseases, most commonly atherosclerosis in the blood and lymphedema/lymphangitis in the lymph. There have been an enormous amount of studies done on blood vasculature in the past few decades, but our understanding of lymphatics in health and disease remains poor. Lymphatics interact systemically and locally with the blood vasculature and we have recently proven that dendritic cells are an important regulator in lymphatic function. Some of these processes can only be proven in a living animal as the interactions and impact of immune cells on vascular flow dynamics and vice versa cannot been mimicked in vitro. Our most recent intravital imaging studies allow the visualization of resident macrophage dynamics within a plaque. Due to the fast movement of cells in the vasculature and the sensitivity of cells to phototoxicity it was challenging to generate a palette of dyes which are low in cytotoxicity able to label the structures and cell populations of interest. It is possible by using fluorophores (Fluorescent proteins and conventional dyes) efficient enough to be excited with low laser power and an emission sufficient enough for being detected. Recently, a femtosecond pulsed Titanium Sapphire (Ti:sap) laser with averaged power ≥ 1W above 1200 nm became available. We have coupled a laser of this type (InSight DS+) together with an conventional Ti:sap-laser (Mai Tai DeepSee) to an customized ultra fast and sensitive scanning Microscope. Using these settings 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.