Development of the bright green and red fluorescent proteins, Clover and mRuby2, creates a fluorescence resonance energy transfer (FRET) pair with the highest Förster radius among existing ratiometric FRET pairs. Substitution of this pair for current FRET pairs in several existing sensors reliably and substantially improves sensor performance. A variety of genetically encoded reporters use changes in fluorescence (or Förster) resonance energy transfer (FRET) to report on biochemical processes in living cells. The standard genetically encoded FRET pair consists of CFPs and YFPs, but many CFP-YFP reporters suffer from low FRET dynamic range, phototoxicity from the CFP excitation light and complex photokinetic events such as reversible photobleaching and photoconversion. We engineered two fluorescent proteins, Clover and mRuby2, which are the brightest green and red fluorescent proteins to date and have the highest Förster radius of any ratiometric FRET pair yet described. Replacement of CFP and YFP with these two proteins in reporters of kinase activity, small GTPase activity and transmembrane voltage significantly improves photostability, FRET dynamic range and emission ratio changes. These improvements enhance detection of transient biochemical events such as neuronal action-potential firing and RhoA activation in growth cones.

Channelrhodopsin 2 (ChR2), a light-activated nonselective cationic channel from Chlamydomonas reinhardtii, has become a useful tool to excite neurons into which it is transfected. The other ChR from Chlamydomonas, ChR1, has attracted less attention because of its proton-selective permeability. By making chimeras of the transmembrane domains of ChR1 and ChR2, combined with site-directed mutagenesis, we developed a ChR variant, named ChEF, that exhibits significantly less inactivation during persistent light stimulation. ChEF undergoes only 33% inactivation, compared with 77% for ChR2. Point mutation of Ile(170) of ChEF to Val (yielding "ChIEF") accelerates the rate of channel closure while retaining reduced inactivation, leading to more consistent responses when stimulated above 25 Hz in both HEK293 cells and cultured hippocampal neurons. In addition, these variants have altered spectral responses, light sensitivity, and channel selectivity. ChEF and ChIEF allow more precise temporal control of depolarization, and can induce action potential trains that more closely resemble natural spiking patterns.