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 silicon-substituted
xanthene, 7-hydroxy-5,5-dimethyl-10-(<i>o</i>-tolyl)dibenzo[<i>b</i>,<i>e</i>]silin-3(5<i>H</i>)-one (<b>2-Me TM</b>), was explored by means absorption, steady-state,
and time-resolved fluorescence. First, the near-neutral pH, ground-state
acidity constant of the dye, p<i>K</i><sub>N‑A</sub>, was determined by absorbance and steady-state fluorescence at very
low buffer concentrations. Next, we determined whether the addition
of phosphate buffer promoted the excited-state proton-transfer (ESPT)
reaction among the neutral and anion form of <b>2-Me TM</b> 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, the
values of the excited-state rate constants were determined by global
analysis of the fluorescence decay traces recorded as a function of
pH, and buffer concentration. 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 <i>pK</i>*<sub>N‑A</sub> and therefore an enhanced photoacid behavior of
the neutral form. Finally, we determined whether <b>2-Me TM</b> 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