Increasingly, fluorescent tools are providing insight into the “dark matter” of the cellular milieu: small molecules, secondary metabolites, metals, and ions. One of the great promises of such tools is the ability to quantify cellular signals in precise locations with high temporal resolution. Yet this is coupled with the challenge of how to ensure that our tools are not perturbing the underlying biology and the need to systematically measure hundreds of individual cells over time. The focus of research in the Palmer Lab is to develop fluorescent tools to illuminate and quantify biochemistry in living cells. In addition to developing such tools, we strive to develop robust systematic analytical approaches for using sensors and fluorescence microscopy to carry out quantitative biochemistry at the single cell level. Over the past 12 years our greatest effort has been in understanding the cell biology of zinc. We have developed fluorescent sensors to map out the spatial distribution of zinc ions and quantify zinc dynamics in live mammalian cells. These tools have allowed us to answer fundamental questions such as: where zinc is located in cells, how much is present in different kinds of cells, under what conditions zinc ions are dynamic, and how the zinc status of healthy cells differs from diseased cells. Along the way, we have also established benchmark analytical approaches for using fluorescent sensors for quantitative biology and biophysics. We have developed microfluidic cell sorting technologies to study and engineer fluorescent tools. We have leveraged our expertise in fluorescent probes, single cell biology and live cell imaging to establish new methods for defining the complex dynamic interface between bacterial pathogens and host mammalian cells. And finally, we have started to expand our reach to develop chemical biology tools for labelling and tracking RNA in live cells. This talk will provide a highlight of these research areas.