Engineering a subcellular targetable, red-emitting, and ratiometric fluorescent probe for Ca2+ and its bioimaging applications

A new, visible-light-excited and red-emitting fluorescent Ca2+ probe, STDBT, was synthesized, which consists of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid as a Ca2+-chelating moiety and two benzothiazolium hemicyanine dyes as fluorophores. The spectral profiles of its free and Ca2+-bound forms were studied. Upon addition of Ca2+, the fluorescence spectra of STDBT displayed a significant enhancement (about 48-fold) in fluorescence intensity and a 20-nm blueshift (from 600 to 580 nm) in the emission spectrum. Both the absorption and the excitation spectra of STDBT showed a very large (more than 100 nm) hypsochromic shift in the long-wavelength maxima upon binding with Ca2+. Interestingly, in contrast with the commonly used Ca2+ indicator Fluo-3, when the acetoxymethyl ester of STDBT enters into cells, it distributes both in the cytosol and the nucleus, but displays a very clear boundary between the two compartments. This allows STDBT to be used as a double targetable Ca2+ probe that can be used to report cytoplasmic Ca2+ and nuclear Ca2+ simultaneously.