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Direct observation of structural heterogeneity and tautomerization of single hypericin molecules
(2021)
Tautomerization is a fundamental chemical reaction which involves the relocation of a proton in the reactants. Studying the optical properties of tautomeric species is challenging because of ensemble averaging. Many molecules, such as porphines, porphycenes, or phenanthroperylene quinones, exhibit a reorientation of the transition dipole moment (TDM) during tautomerization, which can be directly observed in single-molecule experiments. Here, we study single hypericin molecules, which is a prominent phenanthroperylene quinone showing antiviral, antidepressive, and photodynamical properties. Observing abrupt flipping of the image pattern combined with time-dependent density functional theory calculations allows drawing conclusions about the coexistence of four tautomers and their conversion path. This approach allows the unambiguous assignment of a TDM orientation to a specific tautomer and enables the determination of the chemical structure in situ. Our approach can be applied to other molecules showing TDM reorientation during tautomerization, helping to gain a deeper understanding of this important process.
Strong optical mode coupling between two adjacent λ/2 Fabry-Pérot microresonators consisting of three parallel silver mirrors is investigated experimentally and theoretically as a function of their detuning and coupling strength. Mode coupling can be precisely controlled by tuning the mirror spacing of one resonator with respect to the other by piezoelectric actuators. Mode splitting, anti-crossing and asymmetric modal damping are observed and theoretically discussed for the symmetric and antisymmetric supermodes of the coupled system. The spectral profile of the supermodes is obtained from the Fourier transform of the numerically calculated time evolution of the individual resonator modes, taking into account their resonance frequencies, damping and coupling constants, and is in excellent agreement with the experiments. Our microresonator design has potential applications for energy transfer between spatially separated quantum systems in micro optoelectronics and for the emerging field of polaritonic chemistry.
Hypericin is one of the most efficient photosensitizers used in photodynamic tumor therapy (PDT). The reported treatments of this drug reach from antidepressive, antineoplastic, antitumor and antiviral activity. We show that hypericin can be optically detected down to a single molecule at ambient conditions. Hypericin can even be observed inside of a cancer cell, which implies that this drug can be directly used for advanced microscopy techniques (PALM, spt-PALM, or FLIM). Its photostability is large enough to obtain single molecule fluorescence, surface enhanced Raman spectra (SERS), fluorescence lifetime, antibunching, and blinking dynamics. Sudden spectral changes can be associated with a reorientation of the molecule on the particle surface. These properties of hypericin are very sensitive to the local environment. Comparison of DFT calculations with SERS spectra show that both the neutral and deprotonated form of hypericin can be observed on the single molecule and ensemble level.
Hypericin has large potential in modern medicine and exhibits fascinating structural dynamics, such as multiple conformations and tautomerization. However, it is difficult to study individual conformers/tautomers, as they cannot be isolated due to the similarity of their chemical and physical properties. An approach to overcome this difficulty is to combine single molecule experiments with theoretical studies. Time-dependent density functional theory (TD-DFT) calculations reveal that tautomerization of hypericin occurs via a two-step proton transfer with an energy barrier of 1.63 eV, whereas a direct single-step pathway has a large activation energy barrier of 2.42 eV. Tautomerization in hypericin is accompanied by reorientation of the transition dipole moment, which can be directly observed by fluorescence intensity fluctuations. Quantitative tautomerization residence times can be obtained from the autocorrelation of the temporal emission behavior revealing that hypericin stays in the same tautomeric state for several seconds, which can be influenced by the embedding matrix. Furthermore, replacing hydrogen with deuterium further proves that the underlying process is based on tunneling of a proton. In addition, the tautomerization rate can be influenced by a λ/2 Fabry–Pérot microcavity, where the occupation of Raman active vibrations can alter the tunneling rate.
Gold bipyramids (AuBPs) attract significant attention due to the large enhancement of the electric field around their sharp tips and well-defined tunability of their plasmon resonances. Excitation patterns of single AuBPs are recorded using raster-scanning confocal microscopy combined with radially and azimuthally polarized laser beams. Photoluminescence spectra (PL) and excitation patterns of the same AuBPs are acquired with three different excitation wavelengths. The isotropic excitation patterns suggest that the AuBPs are mainly excited by interband transitions with 488/530 nm radiation, while excitation patterns created with a 633 nm laser exhibit a double-lobed shape that indicates a single-dipole excitation process associated with the longitudinal plasmon resonance mode. We are able to determine the three-dimensional orientation of single AuBPs nonperturbatively by comparing experimental patterns with theoretical simulations. The asymmetric patterns show that the AuBPs are lying on the substrate with an out-of-plane tilt angle of around 10–15°.
Monitoring tautomerization of single hypericin molecules in a tunable optical λ/2 microcavity
(2022)
Hypericin tautomerization that involves the migration of the labile protons is believed to be the primary photophysical process relevant to its light-activated antiviral activity. Despite the difficulty in isolating individual tautomers, it can be directly observed in single-molecule experiments. We show that the tautomerization of single hypericin molecules in free space is observed as an abrupt flipping of the image pattern accompanied with fluorescence intensity fluctuations, which are not correlated with lifetime changes. Moreover, the study can be extended to a λ/2 Fabry–Pérot microcavity. The modification of the local photonic environment by a microcavity is well simulated with a theoretical model that shows good agreement with the experimental data. Inside a microcavity, the excited state lifetime and fluorescence intensity of single hypericin molecules are correlated, and a distinct jump of the lifetime and fluorescence intensity reveals the temporal behavior of the tautomerization with high sensitivity and high temporal resolution. The observed changes are also consistent with time-dependent density functional theory calculations. Our approach paves the way to monitor and even control reactions for a wider range of molecules at the single molecule level.
Glioblastoma WHO IV belongs to a group of brain tumors that are still incurable. A promising treatment approach applies photodynamic therapy (PDT) with hypericin as a photosensitizer. To generate a comprehensive understanding of the photosensitizer-tumor interactions, the first part of our study is focused on investigating the distribution and penetration behavior of hypericin in glioma cell spheroids by fluorescence microscopy. In the second part, fluorescence lifetime imaging microscopy (FLIM) was used to correlate fluorescence lifetime (FLT) changes of hypericin to environmental effects inside the spheroids. In this context, 3D tumor spheroids are an excellent model system since they consider 3D cell–cell interactions and the extracellular matrix is similar to tumors in vivo. Our analytical approach considers hypericin as probe molecule for FLIM and as photosensitizer for PDT at the same time, making it possible to directly draw conclusions of the state and location of the drug in a biological system. The knowledge of both state and location of hypericin makes a fundamental understanding of the impact of hypericin PDT in brain tumors possible. Following different incubation conditions, the hypericin distribution in peripheral and central cryosections of the spheroids were analyzed. Both fluorescence microscopy and FLIM revealed a hypericin gradient towards the spheroid core for short incubation periods or small concentrations. On the other hand, a homogeneous hypericin distribution is observed for long incubation times and high concentrations. Especially, the observed FLT change is crucial for the PDT efficiency, since the triplet yield, and hence the O2 activation, is directly proportional to the FLT. Based on the FLT increase inside spheroids, an incubation time 30 min is required to achieve most suitable conditions for an effective PDT.
UV hyperspectral imaging (225 nm–410 nm) was used to identify and quantify the honey- dew content of real cotton samples. Honeydew contamination causes losses of millions of dollars annually. This study presents the implementation and application of UV hyperspectral imaging as a non-destructive, high-resolution, and fast imaging modality. For this novel approach, a reference sample set, which consists of sugar and protein solutions that were adapted to honeydew, was set-up. In total, 21 samples with different amounts of added sugars/proteins were measured to calculate multivariate models at each pixel of a hyperspectral image to predict and classify the amount of sugar and honeydew. The principal component analysis models (PCA) enabled a general differentiation between different concentrations of sugar and honeydew. A partial least squares regression (PLS-R) model was built based on the cotton samples soaked in different sugar and protein concentrations. The result showed a reliable performance with R2cv = 0.80 and low RMSECV = 0.01 g for the valida- tion. The PLS-R reference model was able to predict the honeydew content laterally resolved in grams on real cotton samples for each pixel with light, strong, and very strong honeydew contaminations. Therefore, inline UV hyperspectral imaging combined with chemometric models can be an effective tool in the future for the quality control of industrial processing of cotton fibers.
We study three-color Förster resonance energy transfer (triple FRET) between three spectrally distinct fluorescent dyes, a donor and two acceptors, which are embedded in a single polystyrene nanosphere. The presence of triple FRET energy transfer is confirmed by selective acceptor photobleaching. We show that the fluorescence lifetimes of the three dyes are selectively controlled using the Purcell effect by modulating the radiative rates and relative fluorescence intensities when the nanospheres are embedded in an optical Fabry–Pérot microcavity. The strongest fluorescence intensity enhancement for the second acceptor can be observed as a signature of the FRET process by tuning the microcavity mode to suppress the intermediate dye emission and transfer more energy from donor to the second acceptor. Additionally, we show that the triple FRET process can be modeled by coupled rate equations, which allow to estimate the energy transfer rates between donor and acceptors. This fundamental study has the potential to extend the classical FRET approach for investigating complex systems, e.g., optical energy switching, photovoltaic devices, light-harvesting systems, or in general interactions between more than two constituents.
