## 3. Ratiometric Sensors Constituted by a Single Fluorescent Protein
Subsequently, a modification to r-pHluorin was introduced to enhance the brightness of pHluorin, which was achieved with additional mutations F64L and S65T [30], which are characteristic for EGFP [11]. pHluorin2 is used for pH analysis in human cell lines and yeast _Saccharomyces cerevisiae_[30, 31]. Further, a M153R mutation in the r-pHluorin increases the brightness of e-pHluorinM153R and its _in vivo_ stability, while it does not affect the 410/470 nm excitation ratios at various pH values [32]. The r-pHluorin with mutation A206K named RaVC has been used to study the pH\({}_{\mathrm{i}}\) of the filamentous fungus _Aspergillus niger_[33, 34]. A plant dual-excitation ratiometric pHluorin, PRpHluorin--which is similar to PEpHluorin [4]--is a fusion between a soluble modified GFP (smGFP) [15] with F99S mutation and pHluorin with additional mutations M153T and V163A [4]. The presence of smGFP decreases pK\({}_{\mathrm{a}}\) to 6.6 with no effect on the spectral waveforms of pHluorin. [PERSON] _et al_. [35] introduced four ratiometric sensors named deGFP1 (S65T, H148G, and T203C), deGFP2 (S65T, C48S, and H148C), deGFP3 (S65T and T203C), and deGFP4 (S65T, C48S, H148C, and T203C) that are ratiometric by emission. The deGFPs have a pKa ranging from 6.8 to 8.0. With acidification, with the excitation of deGFPs at 400 nm, a green emission (peak at 515 nm) converts to a blue one with a peak at 460 nm. In addition, the anionic chromophore is directly excited at 480-500 nm, and only a green emission is detected. Therefore, the pH-dependent dual emission is defined at 460 and 515 nm for excitation of a neutral chromophore at 400 nm. The deGFPs are very promising for bioimaging applications, particularly for two-photon excitation. E\({}^{2}\)GFP, a GFP (F64L, S65T, T203Y) variant [36], belongs to the yellow FP class and displays ratiometric characteristics similar to deGFPs [35]. The E\({}^{2}\)GFP is an excellent ratiometric sensor by excitation (e.g., \(\lambda_{\mathrm{x}}\) 458 and 488 nm; \(\lambda_{\mathrm{e}}\) 500-600; pK\({}_{\mathrm{a}}\) 6.9) and by emission (\(\lambda_{\mathrm{x}}\) 458 nm; \(\lambda_{\mathrm{e}}\) 475-525 and 515-600 nm; pK\({}_{\mathrm{a}}\) 7.5). The sensor is quenched by halide ions, but the ratiometric measurements are not affected due to its ratiometric characteristics. Subsequently, E\({}^{1}\)GFP [37] was developed; it is spectroscopically similar to E\({}^{2}\)GFP, but less sensitive to halide ions and with a pK\({}_{\mathrm{a}}\) ranging from 6.4-6.6; therefore, it is more tailored for mildly acidic intracellular compartments. The fluorescence spectra are consistent with the presence of two pH-interconverting forms. Excitation at 408 and 473 nm yields a pH-dependent emission spectra with a peak at 508-510 nm, which is red-shifted (peak at 523 nm) as the pH increases. Further, for live cell imaging, two excitation-wavelength ratiometric setups were used: excitations 488 nm and 458 nm or 488 nm and 406 nm with an emission interval of 500 to 600 nm. The sea cactus _Covernularia obesa_ GFP (CoGFP)--like deGFP and E\({}^{n}\)GFP--has pH-sensitive, ratiometric dual-excitation/emission properties, and dual-color emission maxima upon single-wavelength excitation [38]. The excitation at 388 nm leads to blue fluorescence with a peak at 456 nm at pH 5 and below, and green fluorescence with a peak at 507 nm at pH 7 and above. Excitation at 498 nm triggers green fluorescence with a peak at 507 nm from pH 5-9. Further, fluorescence at 507 nm is ratiometric to that at 456 and 388 nm excitation, with pK\({}_{\mathrm{a}}\) 6.5. Additionalmutations to wtCoGFP generated either brighter fluorescence or different spectral characteristics. It must be noted that only variant-0 has been tested further in live cell imaging. [PERSON] _et al._[39] developed a novel GFP variant isolated from the orange seapen _Ptilosarcus gurneyi_ with good pH responsiveness and excellent dynamic ratio range.