Fluorescence techniques generally offer very high sensitivity allowing observations down to single-molecule level. Additionally, the strong dependence of fluorescence properties on the molecular structure enables the study of small changes involved in chemical reactions and their targeted application.<br/>We have synthesized fluorescent molecules bearing coordinating groups for Cu(II). Its variable oxidation states allow photo-induced electron transfer when brought into close proximity of a chromophore. This non-radiative decay suppresses fluorescence emission and represents a dark state. Single-molecule spectroscopy of Cu-coordinating fluorescent molecules shows switching between bright and dark states which corresponds to binding and unbinding Cu(II). The duration of the bright states directly depends on the Cu-concentration allowing kinetic studies in thermodynamic equilibrium.<br/>To utilize this fluorescence switching we have established a modular synthesis concept to combine two Cu-specific ligands with a large variety of fluorophores from different classes such as rhodamines, oxazines or cyanines. To label cellular structures with regards to fluorescence microscopy we can introduce variable molecular targeting groups like high-affinity biomolecules, click-chemistry moieties and substrates for protein tags. Thereby, these Cu-switches can be used to extend the number of simultaneously observable targets by chemical multiplexing. After labeling two structures with spectrally overlapping fluorophores where one is decorated with a Cu-ligand, we can subsequently image the structures individually. For that we image the first structure in Cu(II)-solution while the second resides in the dark state. After photobleaching, fluorescence of the second structure is restored by removing Cu(II) with EDTA.<br/>Building on that we have recently started to study tetrazine-substituted fluorophores. They undergo electron-inverse Diels-Alder reactions with strained alkenes and alkynes which is why they are of special interest for bioorthogonal labeling. Additionally, the tetrazine group – depending on its position relative to the chromophore – can exhibit fluorescence quenching which is suppressed upon reaction with a dienophile. We now seek to study the process of the Diels-Alder addition and the consecutive reactions by single molecule spectroscopy and to make use of them in sophisticated fluorescence microscopy applications such as no-wash super-resolution microscopy.

From single-molecule studies on chemical reactions to the development of sophisticated labels for bioimaging

256th National Meeting (2018)

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