Real-Time Phosphate Sensing in Living Cells using Fluorescence Lifetime Imaging Microscopy (FLIM)

Phosphate ions play important roles in signal transduction and energy storage in biological systems. However, robust chemical sensors capable of real-time quantification of phosphate anions in live cells have not been developed. The fluorescein derivative dye 9-[1-(2-methyl-4-methoxyphenyl)]-6-hydroxy-3H-xanthen-3-one (2-Me-4-OMe TG) exhibits the characteristic excited-state proton-transfer (ESPT) reaction of xanthenic derivatives at approximately physiological pH resulting in the dependence of the dye’s nanosecond fluorescence decay time on the phosphate buffer concentration. This allows the 2-Me-4-OMe TG dye to be used with fluorescence lifetime imaging microscopy (FLIM) as a real-time phosphate intracellular sensor in cultured cells. This methodology has allowed the time course of cellular differentiation of MC3T3-E1 murine preosteoblast cells to be measured on the basis of the decrease in the decay time of 2-Me-4-OMe TG. These changes were consistent with increased alkaline phosphatase activity in the extracellular medium as a marker of the differentiation process

Auswirkungen des Stabex-Systems auf die Stabilität der Exporterlöse - Eine empirische Analyse zum Nutzen partieller Stabilisierungselemente

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>_N‑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 <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>*_N‑A 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

Visible Absorption and Fluorescence Spectroscopy of Conformationally Constrained, Annulated BODIPY Dyes

Six conformationally restricted BODIPY dyes with fused carbocycles were synthesized to study the effect of conformational mobility on their visible electronic absorption and fluorescence properties. The symmetrically disubstituted compounds (<b>2</b>, <b>6</b>) have bathochromically shifted absorption and fluorescence spectral maxima compared to those of the respective asymmetrically monosubstituted dyes (<b>1</b>, <b>5</b>). Fusion of conjugation extending rings to the α,β-positions of the BODIPY core is an especially effective method for the construction of boron dipyrromethene dyes absorbing and emitting at longer wavelengths. The fluorescence quantum yields Φ of dyes <b>1</b>–<b>6</b> are high (0.7 ≤ Φ ≤ 1.0). The experimental results are backed up by quantum chemical calculations of the lowest electronic excitations in <b>1</b>, <b>2</b>, <b>5</b>, <b>6</b>, and corresponding dyes of related chemical structure but without conformational restriction. The effect of the molecular structure on the visible absorption and fluorescence emission properties of <b>1</b>–<b>6</b> has been examined as a function of solvent by means of the recent, generalized treatment of the solvent effect, proposed by Catalán (<i>J. Phys. Chem. B</i> <b>2009</b>, <i>113</i>, 5951–5960). Solvent polarizability is the primary factor responsible for the small solvent-dependent shifts of the visible absorption and fluorescence emission bands of these dyes

Synchronous Bioimaging of Intracellular pH and Chloride Based on LSS Fluorescent Protein

Ion homeostasis regulates critical physiological processes in the living cell. Intracellular chloride concentration not only contributes in setting the membrane potential of quiescent cells but it also plays a role in modulating the dynamic voltage changes during network activity. Dynamic chloride imaging demands new tools, allowing faster acquisition rates and correct accounting of concomitant pH changes. Joining a long-Stokes-shift red-fluorescent protein to a GFP variant with high sensitivity to pH and chloride, we obtained LSSmClopHensor, a genetically encoded fluorescent biosensor optimized for the simultaneous chloride and pH imaging and requiring only two excitation wavelengths (458 and 488 nm). LSSmClopHensor allowed us to monitor the dynamic changes of intracellular pH and chloride concentration during seizure like discharges in neocortical brain slices. Only cells with tightly controlled resting potential revealed a narrow distribution of chloride concentration peaking at about 5 and 8 mM, in neocortical neurons and SK-N-SH cells, respectively. We thus showed that LSSmClopHensor represents a new versatile tool for studying the dynamics of chloride and proton concentration in living systems