The cyan fluorescent protein family has expanded significantly since the first variant, today notably including the more stable ECFP, brighter Cerulean, or the reef coralderived AmCyan. The complex Catharanthine sulfate fluorescence decay of most CFPs, however, complicates the quantitative interpretation of fluorescence lifetime measurements when it is employed as a FRET donor. To address this issue, mono-exponential cyan variants, such as mTurquoise or the Clavularia coral-derived monomeric teal fluorescent protein were created. We have followed previous work and created a new calcium FRET probe by substituting CFP in TN-L15 with mTFP1, which approximates well to a monoexponential fluorescence decay model and offers a better spectral overlap with the yellow fluorescent protein, being slightly red-shifted compared to the cyan variants. We note that Geiger et al. have previously suggested that a Troponin C-based calcium FRET sensor would benefit from a donor presenting a mono-exponential fluorescence decay profile for fluorescence lifetime readouts. However they did not demonstrate this. To better understand the utility of this new calcium FRET biosensor, called mTFP-TnC-Cit, we have undertaken a protocol that we developed for preparing cytosol preparations from mammalian cells that provides a convenient means to produce bulk solutions of the calcium FRET biosensors. These cytosol preparations provide a biologically relevant system for studying proteins in aqueous phase and can be made more readily and rapidly than bulk solutions based on protein purification from bacterial preparations. The robust data afforded by fluorescence lifetime measurements from solution phase can provide quantitative information on the fraction of molecules undergoing FRET and the distance separating the donor and the acceptor whilst only requiring one additional measurement of the donor fluorescence decay profile in the absence of acceptor. By carrying out a fluorescence lifetime-resolved calcium titration of both FRET biosensors, we have been able to explore the precision of their readouts and measure their dissociation constants. uorescence lifetime data In this set of experiments the donor fluorescence decay from 
TN-L15 and mTFP-TnC-Cit was measured at 12 different free calcium concentrations spanning from negligible calcium up to 40 mM. In the data analysis, two states of the biosensor were considered: one where it is in an “open” conformation, expected to be predominant at low calcium levels and a second, in a closed conformation. For both conformations, the population of donor fluorophores is assumed to have the same number of lifetime components as the free donor fluorophore. Each dataset was fitted using global analysis, thereby allowing the determination of the lifetime components corresponding to each sub-population and their relative contributions at different calcium concentrations. It would be useful to establish the extent to which the fusion of donor fluorophores to the troponin C fragment affects their fluorescence decay profiles. It seems likely that this is a particular issue for CFP – as evidenced by the longer lifetime seen for the open construct compared to CFP alone. This could be important for FLIM microscopy and other applications where relatively broad emission filters are employed. And, as demonstrated by Padilla-Parra et al., the fluorescence lifetime of mTFP1 is much less sensitive to photobleaching than CFP, which can Butenafine hydrochloride undergo photoconversion. Therefore, the reduced sensitivity of mTFP1 to its environment makes it a more robust sensor for quantitative fluorescence lifetime measurements.