Fluorescence-based measurements have become an indispensable part of modern bioanalytical techniques due to their high sensitivity and resolution. Among these, stimulated emission depletion microscopy (STED microscopy) holds a prominent position, as it enables the observation of structures below the diffraction limit of light, thereby extending the possibilities of studying biological systems at the subcellular level. Despite significant technological progress, the main limitation remains the availability of fluorescent probes with properties specifically optimized for the requirements of such methods. Particularly desirable are probes with a large Stokes shift, which reduces the overlap between their excitation and emission spectra and consequently improves image contrast. Moreover, fluorophores with a large Stokes shift enable simultaneous labelling of cells with multiple probes, thereby increasing the amount of information that can be obtained in a single experiment.
Our research focused on the synthesis of four structurally related molecules containing an electrophilic group for cell labelling (Cell Tracker type) based on a coumarin scaffold, which would enable differentiation between various cell lines within a single sample in further studies. We also investigated how selected structural modifications influence the magnitude of the Stokes shift, with the aim of optimizing their photophysical properties and expanding the range of commercially useful probes suitable for STED microscopy.
All four target compounds were successfully synthesized, and their excitation and emission spectra were recorded. Their ability to label living cells and their suitability for visualization using confocal and STED microscopy were evaluated at the Jožef Stefan Institute. The applied synthetic strategies yielded compounds with the desired large Stokes shifts, moderate photostability, and good cell permeability; however, two of the synthesized compounds exhibited cytotoxicity. Regarding labelling selectivity, the compounds were found unsuitable for general cell labelling but effectively stained mitochondria. This observation is not unexpected, as mitochondrial probes are often lipophilic and carry a positive charge. Although the synthesized molecules did not prove to be suitable labels for STED microscopy due to insufficient depletion efficiency, they represent useful tools owing to their large Stokes shifts and serve as valuable starting points for further design and development of more efficient fluorescent probes.
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