## 3. Ratiometric Sensors Constituted by a Single Fluorescent Protein  
The emission intensity of intensity-based pH indicators depends on the total concentration of fluorophore; therefore, it is difficult to determine whether observed changes in fluorescence are due to pH change or indicator concentration. An elegant way to circumvent the problem of the intensity-based pH indicators are ratiometric pH indicators that do not require an independent means of measuring the concentration of protein. Dual excitation ratiometric pH sensors have bimodal excitation spectra, and some also have dual-emission spectra with dose-dependent changes in excitation/emission at increasing acidity (Table 2). These features are an important advantage over the attributes of a single-wavelength FPs, such as e-pHluorin, because they make ratiometric sensors resistant to photo-bleaching and provide variability of indicator loading. \begin{table}
\begin{tabular}{c c c c c c c c} \hline \multirow{2}{*}{**pK**} & \multirow{2}{*}{\(\lambda_{\text{x1}}\) (nm)} & \multirow{2}{*}{\(\lambda_{\text{x2}}\) (nm)} & \multirow{2}{*}{\(\lambda_{\text{e1}}\) (nm)} & \multirow{2}{*}{\(\lambda_{\text{e2}}\) (nm)} & \multirow{2}{*}{**Reference**} \\ \cline{5-6}  & & & \(\varepsilon\) (mM\({}^{-1}\)-cm\({}^{-1}\)) & & & & \\ \hline \multicolumn{6}{c}{pHluorins} \\ \hline R-pHluorin (GenBank: AF058694) & & & & & & \\ (GFP-E132D, S147E, N149L, N164I, K166Q, I167V, R168H, L220F) & 6.9 & 395 & 475 & 508\({}^{\text{a}}\) & \({}^{\text{b}}\) & [13] \\ R-pHluorin2 & & & & & & \\ (R-pHluorin-F64L, Q80R, E132D, S175G) & 7.1 & 395 & 475 & 509\({}^{\text{a}}\) & \({}^{\text{b}}\) & [30,31] \\ R-pHluorin(M153R) & & 7.1 & 395 & 475 & 509\({}^{\text{a}}\) & \({}^{\text{b}}\) & [32] \\ (R-pHluorin-M153R) & & 410 & 470 & 535\({}^{\text{a}}\) & \({}^{\text{b}}\) & [15] \\ PrpHluorin (R-pHluorin for plants) & 6.6 & 395 & 475 & 515\({}^{\text{a}}\) & \({}^{\text{b}}\) & [4] \\ RaVC (R-pHluorin-XX) & 7.1 & 395 & 476 & 508\({}^{\text{a}}\) & \({}^{\text{b}}\) & [33,34] \\ \hline \multicolumn{6}{c}{deGFP} \\ \hline deGFP1 (S65T,Q80R, H148G, T203C) & 8.0 & 400 & 504 & 466 & 516 & [35] \\ deGFP2 (S65T, C48S, Q80R, H148C) & 7.3 & 398 & 496 & 462 & 517 & [35] \\ deGFP3 (S65T, Q80R, T203C) & 6.9 & 396 & 508 & 461 & 518 & [35] \\ deGFP4 & & 7.4 & 400 & 509 & 462 & 518 & [35] \\ (S65T, C48S, Q80R, H148C, T203C) & & & & & & \\ \hline \multicolumn{6}{c}{E\({}^{\text{e}}\)GFP} \\ \hline E\({}^{\text{2}}\)GFP & 6.9–7.5\({}^{\text{a}}\) & 423/401\({}^{\text{d}}\) & 515 & & & \\ (GFP-F64L, S65T, T203Y, L231H) & & _32/23_ & _22_ & & & \\ E\({}^{\text{1}}\)GFP & 6.4–6.6\({}^{\text{a}}\) & 410/400\({}^{\text{d}}\) & 509 & & \\ (GFP-F64L, T203Y) & & _31/29_ & \(5\) & & & \\ \hline \multicolumn{6}{c}{Others} \\ \hline Wt-CoGFP & 6.5\({}^{\text{e}}\) & 388 & 498 & 456/507\({}^{\text{d}}\) & 507 & [38] \\ _Pt_-GFP & 7.3 & 390 & 502 & 508\({}^{\text{a}}\) & \({}^{\text{b}}\) & [39] \\ pHRed (mKeima-A213S) & 7.8; 6.9\({}^{\text{c}}\) & 440 & 585 & 610\({}^{\text{a}}\) & \({}^{\text{b}}\) & [40] \\ SpyHer (HyPer-C199S) & 8.7 & 420 & 490 & 535\({}^{\text{a}}\) & \({}^{\text{b}}\) & [41] \\ \hline \end{tabular} \({}^{\text{a}}\) Valid for both excitations (\(\lambda\)x1 and \(\lambda\)x2); \({}^{\text{b}}\) not determined/not applicable; \({}^{\text{c}}\) pK determined with FLIM; \({}^{\text{d}}\) at pH-5/4 pH\(>\)8, \({}^{\text{e}}\) pK depends on combination of excitation/emission set up.  
\end{table} 
Table 2: Ratiometric pH-sensitive fluorescent proteins. The first study on ratiometric GFP by excitation was that of [PERSON] _et al._[13]. Ratiometric GFP also called ratio-pHluorin (r-pHluorin), and displays an increase in fluorescence at 508 nm after a 475-nm excitation concomitantly with a decrease after a 395-nm excitation upon a pH shift from 7.5 to 5.5, with apparent pK\({}_{\mathrm{a}}\) of 6.9. The functionality of r-pHluorin has been initially tested expressed in _trans_-Golgi network, endosomes, and synapses.