Fluorescent Water-Soluble Polycationic Chitosan Polymers as Markers for Biological 3D Imaging

Over the last decades, various tissue-clearing techniques have broken the ground for the optical imaging of whole organs and whole-organisms, providing complete representative data sets of three-dimensional biological structures. Along with advancements in this field, the development of fluorescent markers for staining vessels and capillaries has offered insights into the complexity of vascular networks and their impact on disease progression. Here we describe the use of a modified water-soluble chitosan linked to cyanine dyes in combination with ethyl cinnamate-based optical tissue clearing for the 3D visualization of tissue vasculature in depth. The water-soluble fluorescent Chitosans have proven to be an optimal candidate for labeling both vessels and capillaries <i>ex vivo</i> thanks to their increased water solubility, high photostability, and optical properties in the near-infrared window. In addition, the nontoxicity of these markers broadens their applicability to <i>in vitro</i> and <i>in vivo</i> biological applications. Despite the availability of other fluorescent molecules for vascular staining, the present study, for the first time, demonstrates the potential of fluorescent chitosans to depict vessels at the capillary level and opens avenues for advancing the diagnostic field by reducing the complexity and costs of the currently available procedures.