, 2009). Moreover, the sigmoidal shape of the voltage/fluorescence response curve of ArcLight indicates that the process is associated with rearrangements arising from gating charge movements and that the

chromophore is not directly affected by changes in the voltage field (like a traditional small molecule organic voltage-sensitive dye). The nonlinearity and slow kinetics of ArcLight check details do not allow detailed studies of action potential shape and propagation within a single cells as is possible with small molecule organic voltage dyes (e.g., Popovic et al., 2011) but do allow action potential detection with lower bandwidth recording. Voltage sensors based on GFP-like fluorescent proteins offer the advantage of substantially greater brightness when compared to other spontaneously fluorescent proteins (Kralj et al., 2011, 2012). While sensors based on microbial rhodopsins have shown promise selleckchem in terms of far red-shifted spectrum and relative response magnitude, the brightness of these probes is dramatically lower than GFP-based probes. Arch D95N has a quantum yield of 0.0004 (Kralj et al., 2012) versus 0.54 for eGFP (Ilagan et al., 2010). In addition the on rate of the nonconducting rhodopsin-based probe (i.e., Arch D95N) is four times

slower than ArcLight probes (41 ms for Arch D94N (Kralj et al., 2012) versus ∼10 ms for the fast component of ArcLight). The large modulatory effect imparted by the D227 mutation introduces the concept of tuning the FP in FP-based voltage sensors as a way to improve them. Previous

studies have made changes to the types of FPs or locations CYTH4 of FPs but have not attempted to modify the FP as a way to improve a probe’s characteristics. In the present study, a small collection of mutations in the FP dramatically increases the change of its fluorescence intensity in response to voltage-induced movements in CiVS. However, these mutations do not alter obvious biophysical properties (i.e., the excitation and emission spectra, pH sensitivity) that would have allowed identification a priori using traditional mutagenesis and screening in E. coli. Mutated sensors still need to be screened in eukaryotic cells in which constructs traffic to the plasma membrane and the resting membrane potential can be set and altered. The ArcLight sensors do not utilize FRET between two fluorescent proteins to produce a signal and it functions at several different insertion sites within the CiVS. ArcLight and its derivatives represent a very substantial improvement in the signal size of a FP voltage sensor, providing a protein-based method to monitor action potentials and subthreshold depolarization in neurons and potentially other cells and organelles.