Direct observation of structural heterogeneity and tautomerization of single hypericin molecules

Tautomerization is a fast chemical reaction where structures of the reactants differ only in the position of a proton and a double bond. Tautomerization often occurs in natural substances and is a fundamental process in organic- and biochemistry. However, studying the optical properties of tautomeric species is challenging due to 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 a single molecule experiment. A prominent phenanthroperylene quinone is hypericin showing antiviral, antidepressive, and photodynamical properties. Here, we study single hypericin molecules by using confocal microscopy combined with higher order laser modes. Observing abrupt flipping of the image pattern allows to draw conclusions about the coexistence of different tautomers and their conversion path. Time-dependent density functional theory calculations show that hypericin is cycling between the four most stable tautomers. This approach allows to unambiguously assign a TDM orientation to a specific tautomer and enables to determine the chemical structure in situ. Additionally, tautomerization can not only be observed by the image pattern orientation, but also as intermittency in the fluorescence emission of a single molecule. Time correlated single photon counting enables to determine the excited state lifetimes of the hypericin tautomers. Our approach is not only limited to hypericin, but can be applied to other molecules showing a TDM reorientation during tautomerization, helping to get a deeper understanding of this important process.