## 6 Instrumentation  
The intrinsic pH of studied subcellular compartments and the type of available equipment determine the selection of FPs for live cell imaging. Intensity-based pH sensors require a collection of data at one wavelength; therefore, rather basic equipment is necessary for one-wavelength intensity measurements. Conversely, a collection of fluorescence at two wavelengths is necessary for dual emission pH sensors; thus, instruments with multiple detectors and filters are needed. Dual excitation sensors, like ratiometric pHluorins, are analyzed with advanced equipment with two excitation sources. Moreover, the spatial analysis of pH within organelles is greatly benefitted with equipment that has a good resolution in Z-direction. Therefore, confocal microscopy is favored over wide-field fluorescent microscopy. The recent developments of highly effective imaging setups with improved resolution and speed, as well as supercontinuum laser have greatly improved the detection of pH in cultured cells and living organisms. A traditional fluorescence spectroscopy is the most extensively applied technique for analyzing the pH\({}_{\text{i}}\) of yeast when pHluorin is used as a pH sensor [51, 52, 53, 54]. Although spectroscopy rapidly records the average pH\({}_{\text{i}}\) of a population, it provides insufficient single-cell information. On the other hand, single-cell analysis offers more detailed insight into population variability, thereby facilitating a considerably deeper understanding of cell physiology. To obtain spatial distribution and a detailed mapping of the pH in single cells, researchers have used pH-sensitive FPs in combination with fluorescence microscopy (Figure 3). Despite the advantages of this approach, the primary drawback of this technique is the need to post-process images for evaluating subpopulation content. Nonetheless, the development of confocal microscopy revolutionized biological sciences and provided great tools for cellular imaging on a subcellular level. Thus, confocal microscopy with excellent resolution (xy 0.2-0.5 \(\upmu\)m and z 0.6-1.5 \(\upmu\)m) provides a very effective way to obtain high-resolution pH maps. Historically, equipment dictated the evolution of fluorescent dyes and FPs. Therefore, cyan, green, and yellow FPs whose excitations correspond to the emission lines of the most widespread Argon laser are best studied (Table 1). Subsequently, microscopes equipped with violet and red laser along with Argon laser promoted the evolution of red- and far red-shifted FPs, as well as ratiometric pH sensors by excitation (Table 2). Advanced confocal microscopes equipped with multiple independent detectors permit simultaneous collection of several fluorescence signals, which agrees with ratiometric sensors by emission (deGFP [35], E\({}^{n}\)GFP [36, 37], Wt-CoGFP [38], and pHusion [47]) and multicolor labeling. Multiphoton microscopy in cases of low photo-toxicity and photo-bleaching is an alternative to single-photon excitation confocal microscopy. The deGFPs and E\({}^{n}\)GFPs have the potential of two-photon ratiometric indicators [35, 36, 37, 48]. Further, fluorescence life time microscopy (FLIM) offers an alternative solution for life cell imaging. pHRed [40] and ECPF [69] exhibit a pH-dependent fluorescence lifetime that can be used to image intracellular pH. Figure 3: **(A)** A set up of dual excitation sequential scanning confocal microscope for imaging ratiometric pHluorin probes. **(B)** Fluorescence excitation spectra of pHluorin in buffers with pH ranging from 5.2 to 8.5. **(C)** A hypothetical pHluorin dose reponse curve; a plot of fluorescence ratio _versus_ pH. The ratios between the emission intensities at 405- and 476-nm are calculated. The physiological target pH range and physiologically relevant dynamic range are indicated. **(D)** Pseudocolored image (right) of permeabilized yeast cells in buffered solution with nigericin is calculated from images taken at 405- and 476-nm excitation (Scans 1 and 2).