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 silicon-substituted xanthene, 7-hydroxy-5,5-dimethyl-10-(o-tolyl)dibenzo[b,e]silin-3(5H)-one (2-Me TM), was explored by means absorption, steady-state, and time-resolved fluorescence. First, the near-neutral pH, ground-state acidity constant of the dye, pKN-A, 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 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, 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 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.

Silicon‐substituted Xanthene Dyes and Their Unique Photophysical Properties for Fluorescent Probes

“…The silicon-substituted xanthened yes are currently utilized not only as scaffolds for fluorescent probes, but also as FRET donors and/ora cceptors, [54] as well as live-cell markers. [55] There are still relatively few fluorophores working in the NIR wavelength region, and the silicon-substituted xanthene dyes are particularly useful choicesf or FRET donors and acceptors because of their biocompatibility.T hese fluorophores are also useful as labeling agents for intracellular proteins [56] andf or small molecules, such as an antimitotic agenta nd metabolites. [57] The use of far-red to NIR fluorophores such as the silicon-substituted xanthenes is experimentallya dvantageous, because they can be excited with limited interference from cellular components (includingf lavin) that contributet ot he significant background autofluorescence observed when GFP is used for imaging.…”

“…The new silicon‐substituted xanthene dyes have already become widely accepted as practical fluorophores working in the far‐red to NIR wavelength regions, and the strategy of replacing oxygen in xanthene dyes with silicon has been extended to other atoms, such as carbon, phosphorus, sulfur, etc. The silicon‐substituted xanthene dyes are currently utilized not only as scaffolds for fluorescent probes, but also as FRET donors and/or acceptors, as well as live‐cell markers . There are still relatively few fluorophores working in the NIR wavelength region, and the silicon‐substituted xanthene dyes are particularly useful choices for FRET donors and acceptors because of their biocompatibility.…”

Synthesis, Photophysics and Solvatochromic Studies of an Aggregated-Induced-Emission Luminogen Useful in Bioimaging

“…Finally, we studied the time of spontaneous incorporation of PEMC into live cells. In contrast to xanthenic dyes [11,22,23,26] (where the incorporation is very fast), PEMC achieves equilibrium approximately 70 minutes after addition of this luminogen. After this period of time, our data show a plateau in the intracellular intensity signal.…”

A Red-Emitting, Multidimensional Sensor for the Simultaneous Cellular Imaging of Biothiols and Phosphate Ions

“…Our research group has recently concentrated efforts on the development of different FLIM-based intracellular sensors [ 14 , 15 ]. Specifically, our thorough studies on the excited-state proton transfer (ESPT) reactions of xanthene derivatives, mediated by the presence of suitable proton acceptor/donor pairs [ 16 , 17 , 18 ], led us to propose a FLIM methodology and a family of sensors for the intracellular quantification of the total phosphate ions concentration [ 19 , 20 ]. The presence of a suitable proton donor/acceptor, such as the pair H 2 PO 4 − and HPO 4 2− , does promote an inter-molecular proton transfer to the prototropic species of xanthene dyes.…”

“…Silicon-substituted rhodamines [ 36 , 37 ], fluoresceins [ 38 , 39 ], and other xanthenes [ 40 ] have been reported in the literature as redshifted fluorophores for bioimaging probes. Interestingly, silicon-substituted fluoresceins, so-called Tokyo magenta (TM) dyes, still maintain the ability of undergoing an ESPT reaction mediated by the presence of the phosphate species present at a near-neutral pH [ 18 ]. A thorough investigation of the photophysics of 7-hydroxy-5,5- dimethyl-10-( o -tolyl)dibenzo[ b,e ]silin-3(5 H )-one (2-Me TM), a dye of the Tokyo magenta family, described the kinetics of the excited-state reaction between the prototropic species of this dye and the H 2 PO 4 − /HPO 4 2− pair as the proton donor/acceptor, reporting the values of all the kinetic rate constants involved in the reaction [ 18 ].…”