The biological functions of plasma membranes remain poorly understood. Fluorescence offers one of the most versatile and powerful approaches for studying biological membranes. It is based on the excitation of fluorophore electrons by light of a specific wavelength, which promotes them to a higher singlet state. After a few nanoseconds, the electrons return to their ground state, emitting energy in the form of a photon, which is observed as fluorescence. Ideal fluorophores for such studies should be photostable, possess a high quantum yield, and have a large molar extinction coefficient. However, effective fluorescent probes for plasma membrane labelling are scarce, and new probes would significantly aid research in this field.
In this master’s thesis, we synthesized two types of fluorescent probes featuring a large Stokes shift for efficient plasma membrane labelling. Both probe types were based on a coumarin fluorophore substituted with a CF3 group at the 4-position. The synthetic strategy included structural modifications to optimize lipophilicity and enhance selectivity for membrane structures. The absorption and emission spectra of the synthesized compounds were recorded. Labelling efficiency of the final compounds in living cells was evaluated at the Jožef Stefan Institute (IJS), along with preliminary testing of selected synthetic intermediates as potential markers for lipid droplets.
Compounds 7, 12, and 16 incorporate two positive charges in their structures and were designed for membrane labelling. Among them, compound 12 demonstrated the best performance, emitting a strong fluorescent signal, with even and stable staining. Due to low signal intensity, photostability and STED microscopy effects for compounds 7 and 16 could not be evaluated.
Compounds 10 and 14, which are synthetic intermediates in the membrane probe synthesis, showed potential for labelling lipid droplets. Both exhibit a large Stokes shift, preferentially localize to lipophilic cellular compartments, and effectively label lipid droplets. They also displayed the highest photostability among all tested compounds. However, none of them exhibited resolution improvement in STED microscopy.
Compound 13 may be suitable for cell tracking applications, as it effectively stains the entire cell interior excluding the nucleus. Nevertheless, it showed cytotoxic effects, as evidenced by morphological changes observed in labelled cells.
We conclude that our work was successful, as we managed to develop potentially useful probes for plasma membrane labeling, as well as one probe for labeling lipid droplets. These compounds are important because they enable the monitoring of morphological and dynamic changes, as well as interactions within the cell.
|
