Abstract
Genetically encoded fluorescent Zn(2+) indicators (GEZIs) are highly attractive research tools for studying Zn(2+) homeostasis and signaling in mammalian cells. Most current GEZIs are based on Förster resonance energy transfer (FRET) between a select pair of fluorescent proteins (FPs) fused with Zn(2+)-binding motifs. One drawback of such FRET-based GEZIs is their broad spectral profile bandwidths, creating challenges when monitoring multiple targets or parameters. To address this issue, we have engineered a group of intensiometric GEZIs based on single teal and red FPs that can be utilized to monitor subcellular Zn(2+) diffusion and glucose-induced Zn(2+) secretion in pancreatic INS-1E β-cells. These GEZIs offer the simplicity of intensiometric measurements, compatibility in multicolor imaging, large dynamic ranges, and relatively small molecular sizes, making them valuable additions to the molecular toolbox for imaging Zn(2+).