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 (k(obs)) for the sulfide-induced deazidation reaction of TFCA in live cells and tissues using TPM. The total sulfide concentration was calculated by using k(obs) = k(2)[HS-], with the k(2) value determined in HEPES/EtOH (1/1, pH = 7.2), and [H2S]/[HS-] = [H+]/K-2. The total sulfide concentration was found to be nearly zero in HeLa cells and 4-7 mu M in rat colon tissues.