On the nature of initial solvation in bulk polar liquids: Gaussian or exponential?

Measurement of time evolution of fluorescence of a probe solute has been a quintessential technique to quantify how dipolar solvent molecules dynamically minimize the free energy of an electronically excited probe. During such solvation dynamics in bulk liquids, a substantial part of relaxation was shown to complete within sub-100 fs from time-gated fluorescence measurements, as also predicted by molecular dynamics simulation studies. However, equivalent quantification of solvation timescales by femtosecond pump-probe and broadband fluorescence measurements revealed an exponential nature of this initial relaxation having quite different timescales. Here, we set out to unveil the reason behind these puzzling contradictions. We introduce a method for estimating probe wavelength-dependent instrument response and demonstrate that the observation of the Gaussian vs exponential nature of initial relaxation is indeed dependent on the method of data analysis. These findings call for further experimental investigation and parallel development of theoretical models to elucidate the molecular-level mechanism accounting for different types of early time solvation.

Separating Vibrational Coherences in Ground/Excited Electronic States of Solvent/Solute Following Non-Resonant/Resonant Impulsive Excitation.

In a quest to track down the origin of coherent vibrational motions observed in femtosecond pump-probe transients, whether they arise from ground/excited electronic state of solute or are contributed by the solvent, we demonstrate a method for extricating vibrations under resonant and non-resonant impulsive excitations using a diatomic solute in condensed phase (iodine in carbon tetrachloride) with aid of spectral dispersion of the chirped broadband probe. Most importantly, we show how a sum over intensities for a select region of detection wavelengths and Fourier transform of data over select temporal window untwine contributions from vibrational modes of different origins. Thus, in a single pump-probe experiment, vibrational features specific to solute as well as solvent are disentangled that are otherwise spectrally overlapping and are non-separable in conventional (spontaneous/stimulated) Raman spectroscopy employing narrowband excitation. We envision wide-ranging applications of this method to unveil vibrational features in complex molecular systems.

A Revisit on Impulsive Stimulated Raman Spectroscopy: Importance of Spectral Dispersion of Chirped Broadband Probe.

The usefulness of a chirped broadband probe and spectral dispersion to obtain Raman spectra under nonresonant/resonant impulsive excitation is revisited. A general methodology is presented that inherently takes care of phasing the time-domain low-frequency oscillations without probe pulse compression and retrieves the absolute phase of the oscillations. As test beds, neat solvents (CCl4, CHCl3, and CH2Cl2) are used. Observation of periodic intensity modulation along detection wavelengths for particular modes is explained using a simple electric field interaction picture. This method is extended to diatomic molecule (iodine) and polyatomic molecules (Nile blue and methylene blue) to assign vibrational frequencies in ground/excited electronic state that are supported by density functional theory calculations. A comparison between frequency-domain and time-domain counterparts, i.e., stimulated Raman scattering and impulsive stimulated Raman scattering using degenerate pump-probe pairs is presented, and most importantly, it is shown how impulsive stimulated Raman scattering using chirped broadband probe retains unique advantages offered by both.

Probing the excited state dynamics of Venus: origin of dual-emission in fluorescent proteins.

Fluorescent proteins exhibit interesting excited state photochemistry, leading to bright fluorescence emission that renders their versatile biological role and wide use as biomarkers. A molecular-level mechanism of the excited state dynamics is desirable to pinpoint the origin of the bright fluorescence of these proteins. Here we present studies on a yellow fluorescent protein variant, Venus, and investigate the photophysics behind the dual fluorescence emission upon UV excitation. Based on our studies, we propose that the unique nature of the potential energy surface is responsible for the observation of minor fluorescence in Venus which is not seen in wild type GFP.

Elucidating photocycle in large Stokes shift red fluorescent proteins: Focus on mKeima.

Large Stokes shift red fluorescent proteins (LSS-RFPs) are genetically encoded and exhibit a significant difference of a few hundreds of nanometers between their excitation and emission peak maxima (i.e., the Stokes shift). These LSS-RFPs (absorbing blue light and emitting red light) feature a unique photocycle responsible for their significant Stokes shift. The photocycle associated with this LSS characteristic in certain RFPs is quite perplexing, hinting at the complex nature of excited-state photophysics. This article provides a brief review on the fundamental mechanisms governing the photocycle of various LSS-RFPs, followed by a discussion on experimental results on mKeima emphasizing its relaxation pathways which garnered attention due to its >200 nm Stokes shift. Corroborating steady-state spectroscopy with computational studies, four different forms of chromophore of mKeima contributing to the cis-trans conformers of the neutral and anionic forms were identified in a recent study. Furthering these findings, in this account a detailed discussion on the photocycle of mKeima, which encompasses sequential excited-state isomerization, proton transfer, and subsequent structural reorganization involving three isomers, leading to an intriguing temperature and pH-dependent dual fluorescence, is explored using broadband femtosecond transient absorption spectroscopy.

Elucidating Contributions from Multiple Species during Photoconversion of Enhanced Green Fluorescent Protein (EGFP) under Ultraviolet Illumination.

Photocycle in wild-type green fluorescent protein (wt-GFP) involves generation of a bright fluorescent deprotonated chromophore from feebly fluorescent protonated form via excited-state proton transfer. In addition to this usual photocycle, wt-GFP is also known to exhibit irreversible photoconversion upon illumination with ultraviolet and visible radiation. However, a detailed understanding of photoconversion in enhanced GFP (EGFP: S65T/F64L mutant of wt-GFP), which predominantly exists in deprotonated form, is yet to be explored. Using 254 nm irradiation, we study how photoconversion proceeds in EGFP. The key findings are observation of spreading out of an isosbestic point and existence of an initial lag phase in spectral kinetics of absorbance, indicative of sequential photoconversion through an intermediate. Fluorescence kinetics of EGFP and its photoproduct are estimated by assigning two unique fluorescence lifetimes which is further complicated by the fact that their fluorescence are spectrally inseparable, as evident from global analysis of fluorescence lifetime. Time-resolved fluorescence anisotropy studies further suggest minimal structural changes in protein scaffold upon photoconversion. Based on these findings, an analytic model is developed to account for the overall decay in fluorescence (as photoconversion proceeds) that inherently incorporates the initial lag phase and a summary of energetics and processes involved is provided.

Planar hexaphyrin-like macrocycles turning into bis-BODIPYs with box-shaped structures exhibiting excitonic coupling.

Planar carbazole based hexaphyrin-like macrocycles with bis-coordinating cores and box-shaped cyclic BODIPYs were synthesized. Solution and solid-state structure analysis of the free macrocycles indicates an inversion of two pyrrole rings, resulting in a two-dipyrrin-like environment. The BF2 complexes show large Stokes shifts and exhibit excitonic coupling, fine-tuned by the meso-substituents.