## 2 Intensity-Based pH Indicators  
The protonated state of the wtGFP absorbs at 395 nm and emits at 508 nm, while the deprotonated state absorbs at 475 nm and emits at 503 nm [9]. The neutral, protonated state is progressively converted to the anionic, deprotonated state as the pH increases. The so-called intensity-based, nonratiometric pH sensors possess good pH responsiveness, but are characterized by very poor emission from the neutral chromophore (395-nm excitation). Due to the rather complex calibration of the intensity-based pH sensors, they are usually used to report changes in pH\({}_{\text{i}}\) rather than pH\({}_{\text{i}}\) itself. Many variations of pH-sensitive FPs have been created with random and targeted mutagenesis of the _Aequorea_ wtGFP [5] or the _Discosoma_ mRFP [10]. These FPs differ in terms of apparent pK\({}_{\text{a}}\) that defines an operational pH range of the sensor and by excitation/emission spectra that delineate the equipment used for analysis (Table 1). [PERSON] _et al._[11] were among the first to explore the possibility that the GFP could be used as a pH sensor in living cells. They synthesized four green GFP mutants with pK\({}_{\text{a}}\) values 6.0 (EGFP, GFP-F64L/ S65T), 5.9 (S65T), 6.1 (Y66H), and 4.8 (T203I). The absorbance and emission spectra of two S65T mutants are similar to that of the wtGFP with two absorption maxima at 390 and 490 nm, with the only excitation wavelength at 490 nm and emission at \(>\)510 nm. To evaluate the suitability of enhanced GFP (EGFP) as a pH sensor in living cells, the EGFP was expressed in cytosol, mitochondria, Golgi, and the endoplasmic reticulum of CHO and LLC-PK1 cells. Two other mutants (Y66H and T203I) have blue-shifted spectra with single maxima and parallel pH-dependent changes in absorbance and fluorescence [11]. In the same year, another blue-shifted pH sensitive protein was synthesized. Extensive mutagenesis of wtGFP generated an enhanced cyan GFP (ECFP); its absorption and emission spectra peak at 440 nm and 480 nm, respectively, with pK\({}_{\text{a}}\) 6.4 [12]. Independent of [PERSON] _et al._, [PERSON] _et al._[13] generated a GFP mutant--termed ecliptic pHluorin--with pKa 7.1. Substitutions S147D, N149Q, T161I, S202F, Q204T, and A206T on wtGFP provoked a reduction in 395 nm excitation and an increase in 475 nm excitation, which is induced by a pH shift. During acidification, the e-pHluorin gradually loses fluorescence. The e-pHluorin is widely used for imaging vesicle fusion events because it is nonfluorescent at a pH lower than 6 at 475 nm excitation, but still weakly seen at 395 nm. A brighter version of the e-pHluorin, a superelliptic pHluorin (e\({}_{\text{s}}\)-pHluorin) [14] contains two additional mutations, F64L and S65T, in the original ecliptic pHluorin, thereby leading to enhanced fluorescence with similar absorption/emission spectra. These two mutations are also characteristic of EGFP [11]. A plant ecliptic pHluorin--PEpHluorin [4]--is a fusion between a soluble-modified GFP (smGFP) [15] with F99S mutation and e-pHluorin with additional mutations M153T and V163A [4]. The presence of smGFP decreases pK\({}_{\text{a}}\) to 6.0 with an acidification-dependent decrease at excitation peaks 395 nm and 475 nm, recorded at 512 nm.