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Published on: July 21, 2018
Photophysical properties of DASPMI as revealed by spectrally resolved fluorescence decays
Radhan Ramadass1, Jürgen Bereiter-Hahn
1Institute for Cell Biology and Neuroscience, Biocenter, JW Goethe University, Max-von-Laue-Strasse 9, D-60439 Frankfurt/Main, Germany. ramadass@bio.uni-frankfurt.de
Abstract:
Photophysical properties of 2-(4-(dimethylamino)styryl)-1-methylpyridinium iodide (DASPMI) in various solvents were investigated using time- and space-correlated single photon counting. DASPMI is known to selectively stain mitochondria in living cells.1,2 The uptake and fluorescence intensity of DASPMI in mitochondria is a dynamic measure of membrane potential. Hence, an endeavor has been made to elucidate the mechanism of DASPMI fluorescence by obtaining spectrally resolved fluorescence decays in different solvents. A biexponential decay model was sufficient to globally describe the wavelength-dependent fluorescence in ethanol and chloroform. While in glycerol, a three-exponential decay model was necessary for global analysis. In the polar low-viscous solvent water, a monoexponential decay model fitted the decay data. The sensitivity of DASPMI to solvent viscosity was analyzed using various proportions of glycerol-ethanol mixtures. The lifetimes were found to increase with increasing solvent viscosity. The negative amplitudes of the short lifetime component found in chloroform and glycerol at the longer wavelengths validated the formation of new excited-state species from the initially excited state. Time-resolved emission spectra in chloroform and glycerol showed a biphasic increase of spectral width and emission maxima. The spectral width had an initial fast increase within 150 ps and a near constant thereafter. A three-state model of generalized scheme, on the basis of successive formation of locally excited state (LE), intramolecular charge transfer state (ICT), and twisted intramolecular charge transfer (TICT) state, has been proposed to explain the excited-state kinetics. The presumed role of solvation dynamics of ICT and TICT states leading to the asymmetrical broadening and structureless fluorescence has been substantiated by the decomposition of time-resolved emission spectra in chloroform, glycerol, and ethanol/glycerol mixtures.
Insights
The photophysical properties of 2-(4-(dimethylamino)styryl)-1-methylpyridinium iodide (DASPMI) were studied in various solvents. DASPMI fluorescence is sensitive to solvent viscosity, with lifetimes increasing with viscosity, and a three-state model explains its excited-state kinetics.
Area of Science:
- Photochemistry
- Biophysical Chemistry
- Spectroscopy
Background:
- 2-(4-(dimethylamino)styryl)-1-methylpyridinium iodide (DASPMI) is a vital fluorescent probe for mitochondria in living cells.
- DASPMI fluorescence intensity and uptake dynamics correlate with mitochondrial membrane potential.
- Understanding DASPMI's photophysical behavior is crucial for accurate cellular analysis.
Purpose of the Study:
- To investigate the photophysical properties of DASPMI in diverse solvents.
- To elucidate the fluorescence mechanism of DASPMI by analyzing spectrally resolved fluorescence decays.
- To determine the influence of solvent viscosity and polarity on DASPMI's excited-state kinetics.
Main Methods:
- Time- and space-correlated single photon counting techniques were employed.
- Spectrally resolved fluorescence decays were measured in various solvents (ethanol, chloroform, glycerol, water).
- Global analysis using mono-, bi-, and tri-exponential decay models was performed.
Main Results:
- DASPMI fluorescence decay kinetics varied with solvent polarity and viscosity, requiring different models (mono-, bi-, tri-exponential).
- Fluorescence lifetimes increased with solvent viscosity, indicating sensitivity to the microenvironment.
- Evidence for new excited-state species formation and time-resolved spectral changes (width, maxima) was observed in specific solvents.
- A three-state model (LE, ICT, TICT) was proposed to explain the observed excited-state kinetics and spectral dynamics.
Conclusions:
- DASPMI's photophysical properties are significantly influenced by solvent characteristics, particularly viscosity and polarity.
- The excited-state behavior of DASPMI can be explained by a model involving locally excited, intramolecular charge transfer, and twisted intramolecular charge transfer states.
- Solvation dynamics play a key role in the observed fluorescence broadening and spectral features, validating the proposed kinetic model.
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