Fluorescence is a physical phenomenon in which fluorophores absorb light of a specific
wavelength and subsequently emit it at a higher wavelength. This process occurs very
rapidly, within the nanosecond range, and has a wide range of applications in science.
Fluorophores, such as coumarins, are commonly used for the selective labelling of cellular
structures, including membranes, lipid droplets, nuclei, etc., allowing their observation under
a fluorescence microscope. An advanced technique in this field is STED microscopy, which
enables the visualization of structures beyond the diffraction limit by precisely controlling
the excitation site.
As part of this master's thesis, we synthesized fluorescent probes for plasma membrane
labelling with a large Stokes shift. Coumarin was used as the core fluorophore, which was
chemically modified to enhance its properties. We investigated how different functional
groups affect the behaviour of these probes in cellular membranes, particularly their ability
to selectively and stably label the plasma membrane. The presence of two positive charges
ensured that the probes remained in the plasma membrane and did not migrate into other
cellular compartments. A large Stokes shift in the selected probes was achieved by
introducing a -CF₃ group at position 4 of the coumarin scaffold. Additionally, for selected
intermediate compounds, we assessed their potential for labelling lipid droplets. The
practical applicability of these probes was tested by cell imaging at the Jožef Stefan Institute.
To ensure the suitability of the selected compounds for STED microscopy, we recorded their
excitation and emission spectra and analyzed their Stokes shift. Based on these results, we
selected compounds with optimal fluorescence properties.
Using STED microscopy, we analyzed compounds 7, 10, 14, and 16. Compounds 10 and 14
exhibited poor selectivity for labelling lipid droplets, whereas compounds 7 and 16 were
effective in labelling the plasma membrane. Among them, compound 16 showed stronger
membrane binding, while compound 7 partially distributed into intracellular structures.
Notably, compound 7 demonstrated high photostability, making it the most suitable for
STED microscopy and positioning it as a promising candidate for further research in super
resolution microscopy.
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