Abstract
Excited state dynamics of deprotonated and protonated fluorescein were investigated by polarization dependent femtosecond time-resolved pump-probe photofragmentation in a 3D ion trap. Transients of deprotonated fluorescein exhibit vibrational wavepacket dynamics with weak polarization dependence. Transients of protonated fluorescein show only effects of molecular alignment and rotational dephasing. The time resolved rotational anisotropy of protonated fluorescein is simulated by the calculated orientational correlation function. The observed differences between deprotonated and protonated fluorescein are ascribed to their different higher lying electronically excited states and corresponding structures. This is partially supported by time-dependent density functional theory calculations of the excited state structures.
Highlights
Fluorescein (FL) and its derivatives represent one of the most popular categories of dyes in biochemistry
Rotational and vibrational wavepacket dynamics were observed for [FL-H]À by velocity-map imaging detection of photoelectrons.22. We extend these experiments by employing the complementary method of linearly polarized femtosecond transient photofragmentation in an ion trap, which allows us to investigate the dynamics of the monoanionic phenolate [FL À H]À and the cationic fluorescein species [FL þ H]þ (Scheme 1)
Irradiation with both pump and probe laser pulses does not lead to other additional fragmentation products but enhances the fragmentation efficiency by a factor of ca. 30 (Fig. S2)45 compared to fragmentation observed at negative delay or by pump-only photoexcitation (Fig. 2). This signal enhancement is a prerequisite for recording transient photofragmentation (tPF) spectra as reported previously23,24,31–33 and contains information on the dynamics of the involved electronically excited states
Summary
Fluorescein (FL) and its derivatives represent one of the most popular categories of dyes in biochemistry. FL is chemically linked to biopolymers and used in (steady-state) fluorescence anisotropy measurements, with the goal to improve microscopic fluorescence imaging and study the dynamics of protein-folding or conformational rearrangements by time-resolved fluorescence anisotropy (TR-FA).. FL is chemically linked to biopolymers and used in (steady-state) fluorescence anisotropy measurements, with the goal to improve microscopic fluorescence imaging and study the dynamics of protein-folding or conformational rearrangements by time-resolved fluorescence anisotropy (TR-FA).12–14 The importance of the latter applications, their perspective for F€orster resonance energy transfer (FRET) studies, and the search for insight into the influence of solvation on the different prototopic FL forms has sparked interest in gas phase studies coupled with mass spectrometric or fluorescence detection.. Due to their high molar absorptivity and fluorescence yield, fluorescein based markers are often used in fluorescence labeling or as probes for, e.g., redox cycles in living cells and sensing of various metals ions or other small molecules. In solution, FL appears pHdependent in up to four prototopic forms (dianion [FL-2H]2À, monoanion [FL À H]À as a phenolate or carboxylate, neutral [FL], and cation [FL þ H]þ) exhibiting specific absorption and emission characteristics. FL is chemically linked to biopolymers and used in (steady-state) fluorescence anisotropy measurements, with the goal to improve microscopic fluorescence imaging and study the dynamics of protein-folding or conformational rearrangements by time-resolved fluorescence anisotropy (TR-FA). The importance of the latter applications, their perspective for F€orster resonance energy transfer (FRET) studies, and the search for insight into the influence of solvation on the different prototopic FL forms has sparked interest in gas phase studies coupled with mass spectrometric or fluorescence detection. In addition, recently, it was convincingly demonstrated that a gas-phase analogue to TR-FA is possible by implementation of a femtosecond time-resolved photodetachment anisotropy (TR-PA) scheme. In that report, rotational and vibrational wavepacket dynamics were observed for [FL-H]À by velocity-map imaging detection of photoelectrons.
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