## 2 Intensity-Based pH Indicators
\begin{table}
\begin{tabular}{c c c c c c c} \hline  & \(\mathbf{pK_{a}}\) & \(\mathbf{\lambda_{\text{n1}}(nm)}\) & \(\mathbf{\lambda_{\text{n2}}(nm)}\) & \(\mathbf{\lambda_{\text{n}}(nm)}\) & \(\mathbf{\lambda_{\text{n}}(nm)}\) & \(\mathbf{\lambda_{\text{n}}(nm)}\) & **Quantum Yield** & **Reference** \\ \hline \multicolumn{6}{c}{Green FP} \\ \hline ECFP (GFP-K26R, F64L, S65T, Y66W, & 6.4 & \({}^{\text{b}}\) & 440 & 480 & 0.40 & [12] \\ N146I, M153T, V163A, N164H, H231L) & & 33 & 3 & & & \\ EGFP & & 396\({}^{\text{a}}\) & 489 & 509 & 0.60 & [11,12] \\ (GFP-F64L, S65T, H231L) & 29 & 60 & & & & \\ Ecliptic pHluorin (GenBank: AF058695) & & & & & \\ (GFP-S147D, N149Q, T161I, V163A, & 7.1 & 396\({}^{\text{a}}\) & 476 & 508 & \({}^{\text{b}}\) & [13] \\ S175G, S202F, Q204T, A206T) & & & & & & \\ Superecliptic pHluorin (GenBank: AY533296) & 7.2 & 396\({}^{\text{a}}\) & 476 & 508 & \({}^{\text{b}}\) & [14] \\ (e-pHluorin-F64L, S65T) & & 395\({}^{\text{a}}\) & 475 & 512 & \({}^{\text{b}}\) & [4] \\ mWasabi & 6.5 & \({}^{\text{b}}\) & 493 & 509 & 0.80 & [16] \\ (GenBank: EU024648) & & 90 & & & & \\ \hline \multicolumn{6}{c}{Yellow FPs} \\ \hline EYFP & 7.1 & 390\({}^{\text{a}}\) & 514 & 527 & 0.61 & [12,17] \\ (GFP-F64L, S65G, S72A, T203Y, H231L) & & 84 & 529 & 0.76 & [18,19] \\ mCitrine(GFP-F64L, S65G, V68L, Q69M, S72A, T203Y, H231L) & 5.7 & \({}^{\text{b}}\) & 516 & 529 & 0.76 & [18,19] \\ mtAlpHi (S65G,V68L, Q69K, S72A, & & & & & \\ T203Y=Camgaroo2 calmodulin & 8.5 & \({}^{\text{b}}\) & 498 & 522 & \({}^{\text{b}}\) & [20] \\ replaced with short aequorin) & & & & & \\ Clover & 6.2 & \({}^{\text{b}}\) & 505 & 515 & 0.76 & [21] \\ \hline \multicolumn{6}{c}{Red FPs} \\ \hline mOrange2 (GenBank: DQ336159) & 6.5 & \({}^{\text{b}}\) & 549 & 565 & 0.60 & [22,23] \\ (mOrange-Q64H, F99Y, E160K, G196D) & & 58 & 565 & 0.60 & [22,23] \\ pHTomato (GenBank: JQ966306) & & & & & \\ (mStrawberry-F41T, Q66T, F83L, & 7.8 & \({}^{\text{b}}\) & 550 & 580 & \({}^{\text{b}}\) & [24] \\ S182K, I194K, V195T, G196D) & & & & & & \\ mNectarine & 6.9 & \({}^{\text{b}}\) & 558 & 578 & \({}^{\text{b}}\) & [25] \\ (GenBank: FJ439505) & & & 588 & & & \\ mKate & 6.2 & \({}^{\text{b}}\) & 588 & 635 & 0.28 & [26] \\ (GenBank: EU383029) & & & 32 & & & \\ mKate2 & 5.4 & \({}^{\text{b}}\) & 588 & & & \\ (GenBank: JB331973) & & & 63 & & & \\ mTangerine & 5.7 & \({}^{\text{b}}\) & 568 & & & \\ (GeneBank: AY678270) & & & 38 & & & \\ mRuby2 & 5.3 & \({}^{\text{b}}\) & 559 & & & \\  & & 43 & & & & \\ \hline \end{tabular} \({}^{\text{a}}\) Poor or no emission at this wavelength excitation; \({}^{\text{b}}\) not determined/not applicable.  
\end{table} 
Table 1: Intensity-based pH-sensitive fluorescent proteins. An enhanced yellow fluorescent protein (EYFP) has a yellow-shifted absorption and fluorophore emission with respect to conventional green FPs [12,17]. This shift in the spectra is the result of a T203Y substitution. Further, EYFP shows an acidification-dependent decrease in the absorbance peak at 514 nm, an emission peak at 527 nm, and a concomitant increase in the absorbance at 390 nm, which is nonfluorescent. An improved yellow FP mCitrine was generated by Q69M and V68L mutations to EYFP. These substitutions reduce sensitivity to chloride ions, improve maturation at 37 \({}^{\circ}\)C, and lower pK\({}_{\mathrm{a}}\) to 5.7 [18,19]. The more acidic pK\({}_{\mathrm{a}}\) classify mCitrine as a pH sensor suited for pH analysis of _medial/trans_-Golgi, as well as secretory and endocytic organelles [12]. [PERSON] _et al._[20] developed a sensor named mtAlpHi for analysis of mitochondrial alkaline pH with an apparent pK\({}_{\mathrm{a}}\) of approximately 8.5. The sensor is composed of EYFP taken from camgaroo-2 [18]. A calmodulin from camgaroo-2 is replaced with aequorin, comprising 73 amino acids that contain second and third EF-hand domains. mtAlpHi undergoes changes only in fluorescent intensity upon pH changes; it is prone to potential artifacts of all nonratiometric indicators. However, the co-transfection with mtECFP largely circumvented this drawback.