Intracellular pH plays an important role in many cellular events such as cell growth, calcium regulation, endocytosis, chemotaxis, cell adhesion and other cellular processes. BCECF has a pKa of ~7.0, which makes spectra of the compound sensitive to pH changes near the physiological range. BCECF fluorescence spectroscopy provides greater sensitivity and convenience for intracellular pH measurement. The dual-excitation fluorescence properties of BCECF with an isosbestic point at 439 nm, makes it an extremely useful excitation-ratiometric pH indicator. These advantages have made BCECF the most widely used fluorescent indicator for measuring intracellular pH. However, all the commercial BCECF AM is a complex mixture of at least three isomers with different ratios from batch to batch, complicating the BCECF applications. BCBCF is developed to overcome this isomer difficulty associated with BCECF AM. As BCECF, BCBCF exhibits a pH-dependent dual excitation, essentially identical to BCECF. It has pKa of ~7.0, identical to BCECF too. As with BCECF, the dual excitation spectrum of BCBCF with an isosbestic point at 454 nm should make BCBCF a good excitation-ratiometrie pH indicator. BCBCF ratiometric imaging makes intracellular pH determination essentially independent of several variable factors, including dye concentration, path length, cellular leakage and photobleaching rate. BCBCF, AM is a single isomer, making the pH measurement much more reproducible than the BCECF, AM, which is consisted of quite a few different isomers.
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