Choosing the Right Fluorescent Probe

Fluorescence microscopy and spectroscopy are by now used routinely in any laboratory working on the field of basic and applied biological sciences. A wide and expanding library of small organic fluorophores with radically different properties has been made available, offering great flexibility to the user of fluorescence-based methods. Beyond small organic fluorophores, the development of fluorescent proteins allowed for the introduction of genetically encoded fluorescence tagging, a novel tool that quickly revolutionized cellular imaging and cell biology. Still, a considerable fraction of casual users of fluorescence tools do not follow rational considerations when selecting a fluorescent probe for their intended application, often relying on trial and error alone, which inevitably leads to a decrease in data quality and limits the potential of fluorescence-based methods. This chapter aims to present an overview of the most important considerations to be made when selecting a fluorescent reporter. Fluorophore properties and their importance for fluorescence assays will be discussed. A list of different fluorophores and a summary of their properties will also be presented as a tool to assist in the process of choosing the right fluorescence probe. Finally, specific applications such as super-resolution microscopy require very specific fluorophores, and these will be discussed separately.