Fluorescence is a type of luminescence in which a fluorophore absorbs light of a specific wavelength, causing the fluorophore to transition to an excited state. After a certain period of time (a few nanoseconds), the fluorophore emits a photon and spontaneously returns to its ground state. This phenomenon is exploited in fluorescence microscopy, which is an indispensable tool in virtually all biosciences. An advancement of fluorescence microscopy is Stimulated Emission Depletion (STED) microscopy, which allows us to observe much smaller structures of nanometer sizes. The fluorophores used for the super-resolution technique STED have specific properties; they must be photostable, possess the appropriate absorption and emission spectra, and specifically label the structures we want to observe.
The purpose of this master's thesis is to present the synthesis of new merocyanines (coumarin derivatives) with the aim of using structural changes to convert them into effective markers for lipid droplets and to improve their properties for STED microscopy. Some such markers have already been synthesized at the Faculty of Pharmacy, but the yields were low, partly due to the formation of side products. Additionally, we aimed to investigate how different electron-withdrawing and electron-donating groups affect the photophysical properties of the designed probes. Therefore, we pursued a different synthesis route and synthesized 8 new fluorescent probes, for which we measured the excitation and emission spectra. Five of the prepared probes (7, 9, 11, 12, and 14) exhibited suitable spectral properties for further experiments on the STED microscope at the Jozef Stefan Institute (IJS).
We incubated cells from the LA-4 cell line with the prepared probes. Based on the labeling properties of similar probes, we expected our probes to label lipid droplets. These are relatively understudied cellular organelles, yet they are undoubtedly implicated in the development of certain cancerous and infectious diseases.
After experiments on the STED microscope, we found that the probes did not exhibit cytotoxicity, and probe 11 stood out in terms of photostability, as its signal even increased with exposure to light. However, none of the probes satisfactorily labeled lipid droplets, or they labeled organelles within the cell nonspecifically.
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