Photophysics of a Live-Cell-Marker, Red Silicon-Substituted Xanthene Dye

Abstract

Dyes with near-red emission are of great interest because of their undoubted advantages for use as probes in living cells. In-depth knowledge of their photophysics is essential for employment of such dyes. In this article, the photophysical behavior of a new siliconsubstituted xanthene, 7-hydroxy-5,5-dimethyl-10-(o-tolyl)dibenzo[b,e]silin-3(5H)-one (2-Me TM), was explored using absorption, steady-state, and time-resolved fluorescence. First, the near-neutral pH, ground state acidity constant of the dye, pK*N-A, was determined by both absorbance and steady-state fluorescence methods in the presence of very low buffer concentrations. Next, we determined whether the addition of phosphate buffer promoted the excited-state proton exchange reaction between the neutral and anion form of 2-Me TM in aqueous solutions at near-neutral pH. For this analysis, both the steady-state fluorescence method and time-resolved emission spectroscopy (TRES) were employed. The TRES experiments demonstrated a remarkably favored conversion of the neutral form to the anion form. Then, fluorescence decay traces recorded as a function of buffer concentration and pH were globally analyzed to determine the values of the excited-state rate constants. The revealed kinetic parameters were consistent with the TRES results, exhibiting a higher rate constant for deprotonation than for protonation, which implies an unusual low value of the excited-state acidity constant pK*N-A and therefore an enhanced photoacid behavior of the neutral form. Finally, we determined whether 2-Me TM could be used as a sensor inside live cells by measuring the intensity profile of the probe in different cellular compartments of HeLa 229 cells.