Investigation of the natural world relies on our ability to perceive it, and the conclusions we draw are limited by what we can detect. The development of new tools to probe and explore the cellular world require commitment, perseverance, and new approaches to research. This thesis focuses on the development of a new biochemical tool capable of attaching GFP-based fluorescent biosensors to the glycocalyces of cell surfaces to visualize the flux of ions and metabolites out of living cells. Chapter I addresses the strengths and limitations of current methods to investigating large-pore hemichannels, including the approach developed here. Chapter II elucidates the design, development, synthesis, and characterization of a novel organic compound, Ni2+-tris-NTA-PEG4-DBCO, that is capable of both covalently modifying the extracellular sugar coating of mammalian cells, and binding oligohistidine-tagged fluorescent biosensors. This unique approach to “biosensor repurposing” places ion and metabolite biosensors at the source of release. We demonstrate this approach to visualize glutamate and protons at the cell surface, and the numerous cell types capable of further investigation. Chapter III focuses on the first known attempt to visualize endogenous ATP release from large-pore hemichannels using fluorescent biosensors. Here, pannexin- 1 and CALHM1 hemichannels were overexpressed in HEK293T and HeLa cells, respectively, leading to ATP release. We attempted to fluorescently visualize these release events in real-time at the cell surface. Chapter IV concludes with future directions.