Investigating Two-Photon-Induced Fluorescence in Rhodamine-6G in Presence of Cetyl-Trimethyl-Ammonium-Bromide.

We investigate the effect of cetyl-trimethyl-ammonium-bromides (CTAB) concentration on the fluorescence of Rhodamine-6G in water. This spectroscopic study of Rhodamine-6G in presence of CTAB was performed using two-photon-induced-fluorescence at 780 nm wavelength using high repetition rate femtosecond laser pulses. We report an increment of ∼10 % in the fluorescence in accordance with ∼12 % enhancement in the absorption intensity of the dye molecule around the critical micellar concentration. We discuss the possible mechanism for the enhancement in the two-photon fluorescence intensity and the importance of critical micellar concentration.

Cyclodextrin insulation prevents static quenching of conjugated polymer fluorescence at the single molecule level.

Conjugated polymers (CPs) are promising materials for fluorescence imaging application. However, a significant problem in this field is the unexplained abnormally low fluorescence brightness (or number of fluorescence photons detected per one excitation photon) exhibited by most of CP single chains in solid polymer hosts. Here it is shown that this detrimental effect can be fully avoided for short chains of polyfluorene-bis-vinylphenylene (PFBV) embedded in a host polymer matrix of PMMA, if the conjugated backbone is insulated by cyclodextrin rings to form a polyrotaxane (PFBV-Rtx). Fluorescence kinetics and quantum yields are measured for the polymers in liquid solutions, pristine films, and solid PMMA blends. The fluorescence brightness of PFBV-Rtx single chains dispersed in a solid PMMA is very close to that expected for a chain with 100% fluorescence quantum yield, while the unprotected PFBV chains of the same length possess 4 times lower brightness. Despite this, the fluorescence decay kinetics are the same for both polymers, suggesting the presence of static or ultrafast fluorescence quenching in the unprotected polymer. About 80% of an unprotected PFBV chain is estimated to be completely quenched. The hypothesis is that the cyclodextrin rings prevent the quenching by working as 'bumpers' reducing the mechanical forces applied by the host polymer to the conjugated backbone and help retaining its conformational freedom. While providing a recipe for making CP fluorescence bright at the single-molecule level, these results identify a lack of fundamental understanding in the community of the influence of the environment on excited states in conjugated materials.

Evidence of excited state localization and static disorder in LH2 investigated by 2D-polarization single-molecule imaging at room temperature.

Two-dimensional polarization fluorescence imaging of single light harvesting complexes 2 (LH2) of Rps. acidophila was carried out to investigate the polarization properties of excitation and fluorescence emission simultaneously, at room temperature. In two separate experiments we excited LH2 with a spectrally narrow laser line matched to the absorption bands of the two chromophore rings, B800 and B850, thereby indirectly and directly triggering fluorescence of the B850 exciton state. A correlation analysis of the polarization modulation depths in excitation and emission for a large number of single complexes was performed. Our results show, in comparison to B800, that the B850 ring is a more isotropic absorber due to the excitonic nature of its excited states. At the same time, we observed a strong tendency for LH2 to emit with dipolar character, from which preferential localization of the emissive exciton, stable for minutes, is inferred. We argue that the observed effects can consistently be explained by static energetic disorder and/or deformation of the complex, with possible involvement of exciton self-trapping.

Hydrostatic High-Pressure-Induced Denaturation of LH2 Membrane Proteins.

The denaturation of globular proteins by high pressure is frequently associated with the release of internal voids and/or the exposure of the hydrophobic protein interior to a polar aqueous solvent. Similar evidence with respect to membrane proteins is not available. Here, we investigate the impact of hydrostatic pressures reaching 12 kbar on light-harvesting 2 integral membrane complexes of purple photosynthetic bacteria using two types of innate chromophores in separate strategic locations: bacteriochlorophyll-a in the hydrophobic interior and tryptophan at both protein-solvent interfacial gateways to internal voids. The complexes from mutant Rhodobacter sphaeroides with low resilience against pressure were considered in parallel with the naturally robust complexes of Thermochromatium tepidum. In the former case, a firm correlation was established between the abrupt blue shift of the bacteriochlorophyll-a exciton absorption, a known indicator of the breakage of tertiary structure pigment-protein hydrogen bonds, and the quenching of tryptophan fluorescence, a supposed result of further protein solvation. No such effects were observed in the reference complex. While these data may be naively taken as supporting evidence of the governing role of hydration, the analysis of atomistic model structures of the complexes confirmed the critical part of the structure in the pressure-induced denaturation of the membrane proteins studied.

Protein Configuration Landscape Fluctuations Revealed by Exciton Transition Polarizations in Single Light Harvesting Complexes.

Protein is a flexible material with broad distribution of conformations forming an energy landscape of quasi-stationary states. Disentangling the system dynamics along this landscape is the key for understanding the functioning of the protein. Here we studied a photosynthetic antenna pigment-protein complex LH2 with single molecule two-dimensional polarization imaging. Modeling based on the Redfield relaxation theory well describes the observed polarization properties of LH2 fluorescence and fluorescence excitation, strongly suggesting that at 77 K the conformational subspace of the LH2 is limited to about three configurations with relatively frequent switching among each other. At room temperature the next level of fluctuations determines the conformational dynamics. The results support the multitier model of the energy landscape of proteins and demonstrate the potential of the method for the studies of structural dynamics in proteins.

Ultrafast Dynamics of a Green Fluorescent Protein Chromophore Analogue: Competition between Excited-State Proton Transfer and Torsional Relaxation.

The competition between excited-state proton transfer (ESPT) and torsion plays a central role in the photophysics of fluorescent proteins of the green fluorescent protein (GFP) family and their chromophores. Here, it was investigated in a single GFP chromophore analogue bearing o-hydroxy and p-diethylamino substituents, OHIM. The light-induced dynamics of OHIM was studied by femtosecond transient absorption spectroscopy, at different pH. We found that the photophysics of OHIM is determined by the electron-donating character of the diethylamino group: torsional relaxation dominates when the diethylamino group is neutral, whereas ultrafast ESPT followed by cis/trans isomerization and ground-state reprotonation are observed when the diethylamino group is protonated and therefore inactive as an electron donor.

Real-time monitoring of chromophore isomerization and deprotonation during the photoactivation of the fluorescent protein Dronpa.

Dronpa is a photochromic green fluorescent protein (GFP) homologue used as a probe in super-resolution microscopy. It is known that the photochromic reaction involves cis/trans isomerization of the chromophore and protonation/deprotonation of its phenol group, but the sequence in time of the two steps and their characteristic time scales are still the subject of much debate. We report here a comprehensive UV-visible transient absorption spectroscopy study of the photoactivation mechanism of Dronpa, covering all relevant time scales from ∼100 fs to milliseconds. The Dronpa-2 variant was also studied and showed the same behavior. By carefully controlling the excitation energy to avoid multiphoton processes, we could measure both the spectrum and the anisotropy of the first photoactivation intermediate. We show that the observed few nanometer blue-shift of this intermediate is characteristic for a neutral cis chromophore, and that its anisotropy of ∼0.2 is in good agreement with the reorientation of the transition dipole moment expected upon isomerization. These data constitute the first clear evidence that trans → cis isomerization of the chromophore precedes its deprotonation and occurs on the picosecond time scale, concomitantly to the excited-state decay. We found the deprotonation step to follow in ∼10 µs and lead directly from the neutral cis intermediate to the final state.