## 7 Conclusions  
Undoubtedly, the maintenance of an appropriate pH within individual membrane-enclosed compartments is important for normal physiology of the cell and organelles. Over the previous decade, considerable progress has been made in understanding the pH regulatory mechanisms of the cell that progress in tandem with recent developments of highly effective imaging setups and genetically encoded pH-sensitive fluorescent sensors. In merely fifteen years, we have witnessed an expansion in  
Figure 4: Flow cytometry for high-throughput analysis of cytosolic pH in cells expressing ratiometric pH sensors by excitation [31]. (**A**) The light paths of excitation at 405 and 488 nm and the emission optics are illustrated. Fluorescence intensity data are depicted for both detection channels—F(488 nm) and F(405 nm). (**B**) A 2D dot plot of F405-nm _versus_ F488-nm shows grouped fluorescence signals. (**C**) The ratio of the fluorescence intensities of F(405 nm)/F(488 nm) plotted as the ratio height signal generates a narrow peak. (**D**) An overlay of 2D dot plots of F(405 nm) _versus_ F(488 nm) for pH sensor at indicated pH values. (**E**) An overlay of ratios of the fluorescence intensities of F(405 nm)/F(488 nm) depicted from (**F**). Note: In less than 2 min, the fluorescence signals of 50,000 cells can be analyzed and post-processed.  
genetically encoded pH-sensitive fluorescent proteins. pH sensors span almost the entire visible spectrum from green to red and with pKa that extends over the entire physiological pH range. Most modern FPs have been modified by random and/or iterative mutagenesis to optimize, improve, or enhance fluorescence characteristics and their stability as proteins. The sensors for virtually any subcellular location have been engineered from only a few building blocks. The interplay of sophisticated imaging techniques and transgene pH sensors has provided a very effective method for obtaining spatial and temporal high-resolution pH maps and the ability to monitor signaling dynamics on single-cell and subcellular levels.  
In this review, we have described pH-sensitive fluorescent proteins that we consider to be useful or as having potential for generating novel sensors for live cell applications. The trend in the evolution of pH sensors is oriented toward generating brighter, photostable yellow and red ratiometric FPs to limit the cell damage that might be caused by the illumination of the fluorophore. Advances have been made for pH-sensitive FPs with narrow excitation and emission peaks to suit multicolor labeling. The narrow spectral peaks minimize the crosstalk between fluorophores, thereby providing more accurate results in multicolor analysis. With regard to developments in equipment, pH sensors that are suited for fluorescence lifetime microscopy and two-photon laser scanning fluorescence microscopy have been engineered. Transgene pH sensors are already changing the boundaries of our understanding of _in vivo_ cellular pH dynamics at scales from the subcellular to the entire organism. Further, pH-sensitive FPs expressed in the cell and subcellular compartments provide the most physiologically relevant information on the spatial and temporal behavior of pH homeostasis. Overall, we anticipate that this overview will help in deciding which pH-sensitive fluorescent protein should be selected for forthcoming cellular imaging experiments, and how the selected pH sensor could be applied to obtain accurate answers to various questions in this regard.  
This research was supported by the Slovenian Research Agency and EN-FIST, Centre of Excellence.