The development of fluorescent proteins (FPs) and a detailed understanding of their photophysics is critical for quantitative bioimaging. I will describe our research to improve the brightness of red FPs. The molecular brightness is based on the product of the extinction coefficient and fluorescence quantum yield (QY). Development of brighter FPs has focused on increasing the QY, which is directly proportional to fluorescence lifetime (FL) in this system. We performed directed evolution of red FPs with random and site-directed mutant libraries on a microfluidic sorting system capable of FL and brightness selections. We also applied site directed mutagenesis to investigate the effects of molecular interactions on FL and QY. We selected mutants derived from two families of red FPs, showing that an improvement in FL brings about an expected linear increase in QY (~3.2 fold for mCherry and ~2.3 fold for FusionRed) due to significant decreases in their non-radiative rates. This strategy is the basis of a productive route for engineering brighter FPs.
Ralph Jimenez earned his PhD in Physical Chemistry at the University of Chicago and was a postdoc at UC San Diego and a Senior Research Associate at the Scripps Research Institute before joining JILA in 2003. His research on the directed evolution of fluorescent proteins combines protein engineering with microfluidics technologies and ultrafast spectroscopy. Recently, his group has been exploring the potential for quantum measurement techniques to enhance the sensitivity and information content of molecular spectroscopy and imaging.