Quantitative estimation of the total sulfide concentration in live tissues by two-photon microscopy.

Hydrogen sulfide (H2S) is a newly recognized transmitter, which protects various organs from oxidative stress. In this article, we report a ratiometric two-photon probe, TFCA, which can be excited by 750 nm femtosecond pulses, shows a 110-fold increase in the intensity ratio upon reaction with HS(-) and high selectivity for HS(-) and can visualize the total sulfide ([H2S] + [HS(-)]) distribution in live tissue by two-photon microscopy (TPM). We also developed a kinetic method to quantitatively estimate the total sulfide concentration ([H2S] + [HS(-)]) in live tissues. The kinetic method allowed us to measure the observed rate constants (kobs) for the sulfide-induced deazidation reaction of TFCA in live cells and tissues using TPM. The total sulfide concentration was calculated by using kobs = k2[HS(-)], with the k2 value determined in HEPES/EtOH (1/1, pH = 7.2), and [H2S]/[HS(-)] = [H(+)]/Ka. The total sulfide concentration was found to be nearly zero in HeLa cells and 4-7 μM in rat colon tissues.