pHimaging

pH imaging refers to techniques that visualize and quantify the distribution of pH values within cells, tissues, or organisms. It relies on fluorescent or luminescent probes whose optical properties change with proton concentration, enabling spatial maps and, in some cases, temporal dynamics of pH.

Common approaches include chemical fluorescent dyes such as BCECF, SNARF, and FITC-based indicators that are either cell-permeant or localized to organelles. Many dyes are used in a ratiometric format, measuring emission at two wavelengths to cancel out dye concentration and path length. Genetically encoded pH sensors, such as pH-sensitive GFP variants (e.g., pHluorin) targeted to specific compartments, allow measurement of pH in living cells with genetic specificity. Fluorescence lifetime imaging (FLIM) can be used when lifetime changes correlate with pH. Calibration is required, typically by equilibrating intracellular or extracellular pH using ionophores like nigericin in high potassium buffers.

Applications include monitoring cytosolic, mitochondrial, lysosomal, and extracellular pH, studying organelle function, metabolic states, autophagy, endocytosis, and pathology in cancer or neurodegenerative diseases. In vivo pH imaging uses near-infrared probes to map tumor acidity or tissue pH dynamics.

Limitations include narrow pH sensitivity ranges, probe buffering effects, photobleaching, dye loading variability, compartmentalization, and potential interference with cellular processes. Accurate interpretation requires careful calibration, controls, and, for in vivo work, consideration of tissue light scattering and autofluorescence.

Overall, pH imaging provides spatial and, with time-resolved methods, dynamic information on proton distribution, contributing to studies of physiology and disease.