Abstract
Fluorescence imaging represents cornerstone technology for studying biological function at the cellular and molecular levels. The technology's centerpiece is a prolific collection of genetic reporters based on the green fluorescent protein (GFP) and related analogs. More than two decades of protein engineering have endowed the GFP repertoire with an incredible assortment of fluorescent proteins, allowing scientists immense latitude in choosing reporters tailored to various cellular and environmental contexts. Nevertheless, GFP and derivative reporters have specific limitations that hinder their unrestricted use for molecular imaging. These challenges have inspired the development of new reporter proteins and imaging mechanisms. Here, we review how these developments are expanding the frontiers of reporter gene techniques to enable nondestructive studies of cell function in anaerobic environments and deep inside intact animals-two important biological contexts that are fundamentally incompatible with the use of GFP-based reporters.