Organelle‐targeting, red‐emitting fluorescent probes with superior photo‐physical properties for multiplexing with green fluorescent protein (GFP) in live cells

Cell‐based assays are increasingly gaining in popularity in the biotechnology and pharmaceutical industry due to their high physiological relevance. As trends advance toward analysis of living biological systems, fluorescent proteins (such as GFPs) have become indispensable tools for live‐cell analysis. One important application of GFPs is fluorescence co‐localization imaging, a powerful approach for exploring the targeting of molecules to intracellular compartments and for screening of their associations and interactions. To date, however, concerted efforts in developing fluorescent organelle‐targeting probes, specifically tailored to working in concert with GFPs, has been limited. Photo‐conversion of red‐fluorescent dyes to fluoresce green and metachromatic artifacts wherein fluorescent dyes emit both red and green lead to spurious results in GFP co‐localization experiments. Additionally, many organelle‐targeting probes photo‐bleach rapidly, are subject to quenching upon concentration in organelles or only transiently associate with the target organelle, requiring imaging within a minute of dye addition. Consequently, a new family of red‐emitting, cell‐permeable probes that spontaneously localize to specific subcellular organelles was developed. It is demonstrated that the probes can be readily used in combination with other common UV‐ and visible‐light excitable organic fluorescent dyes and various fluorescent proteins in multi‐color imaging and detection applications. It is further shown that the probes emit in the Texas Red region of the visible spectrum, and are highly resistant to photo‐bleaching, concentration quenching and photo‐conversion.