Super-resolution microscopy that enables us to observe structures smaller than the diffraction limit, has thoroughly improved the understanding of cellular and molecular biology. Stimulated emission depletion microscopy (STED) is one of the super-resolution microscopy methods, which enables us the observation of subcellular structures, the studying of the dynamic of living cells on nanoscale and this way enables us an insight into the actions, behaviour and interactions, which cannot be observed in fixated cells. Fluorescent dyes used for labelling structures must meet different criteria in order for them to be considered as appropriate for the STED microscopy. For the selective labelling of plasma membrane, the probes must have other characteristics, as well, so the choice is even smaller.
In our work we synthesized and analysed probes for the selective labelling of plasma membranes based on the quinoline scaffold and coumarin probe MePyr500, which has excellent photophysical properties, but unspecifically labels all the membranes of the cell. We modified the probe with the addition of an extra charge, which would prevent partitioning of the probe into the intracellular membranes. We analysed prepared fluorophores with spectroscopic methods and measured their excitation and emission spectrums. We wanted the synthesized dyes to be bright, stable, with a large Stokes shift and at the same time to specifically label only plasma membrane. The most suitable probes were sent to the Institute Jožef Stefan, where they stained biological samples and evaluated their photophysical properties when used in STED microscopy.
There were no problems with the planned compounds as far as the synthesis went. The most successful was the coumarin probe SHE 2n, which labels outer membrane of the cell in a specific way due to the two positive charges and is also very photostable. It enables the production of super-resolution pictures and it is not cytotoxic. It therefore makes sense to synthesize its analogs and to optimise its reaction conditions. Synthesized analogs of quinolone probes such as SHE 1h are not suitable for STED microscopy since they lack significant emission at STED laser wavelengths (775 nm) and therefore do not enable the production of super-resolution images. Quinoline probes also do not label plasma membrane selectively.
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