Sinteza in vrednotenje fluorescenčnih sond z velikim Stokesovim premikom za označevanje plazemske membrane

Poslek Baršić, Lea (avtor), Pajk, Stane (mentor), Kokot, Hana (komentor)

Fluorescenca je fizikalni pojav, pri katerem fluorofori absorbirajo svetlobo določene valovne dolžine in jo nato oddajo pri višji valovni dolžini. Proces poteka zelo hitro, v območju nanosekund, in ima širok spekter uporabnosti v znanosti. Fluorofori, kot so kumarini, se pogosto uporabljajo za selektivno označevanje celičnih struktur, kot so membrane, lipidne kapljice, jedra itd., da jih lahko opazujemo s fluorescenčno mikroskopijo. Nadgradnja slednje je STED mikroskopija, ki omogoča opazovanje struktur z ločljivostjo pod difrakcijsko mejo s pomočjo natančne regulacije mesta vzbujanja. V okviru magistrske naloge smo sintetizirali fluorescenčne sonde z velikim Stokesovim premikom za označevanje plazemske membrane. Kot osnovni fluorofor smo uporabili kumarin, ki smo ga kemijsko modificirali za izboljšanje njegovih lastnosti. Raziskovali smo, kako različne funkcionalne skupine vplivajo na obnašanje teh sond v celičnih membranah, predvsem, kako selektivno in stabilno označujejo plazemsko membrano. Prisotnost dveh pozitivnih nabojev je omogočila, da se sonde zadržujejo v plazemski membrani in ne prehajajo v druge celične predele. Velik Stokesov premik pri izbranih sondah je bil dosežen z uvedbo -CF3 skupine na mestu 4 kumarinskega skeleta. Poleg tega smo pri izbranih spojinah vmesnih stopenj reakcij preverili njihovo uporabnost tudi za označevanje lipidnih kapljic. Praktična uporabnost teh sond je bila preskušena z mikroskopiranjem celic na Institutu Jožef Stefan. Da bi zagotovili ustreznost izbranih spojin za mikroskopijo STED, smo posneli njihove ekscitacijske in emisijske spektre ter analizirali njihov Stokesov premik. Na podlagi teh rezultatov smo izbrali spojine z optimalnimi fluorescenčnimi lastnostmi. Z uporabo STED mikroskopije smo analizirali spojine 7, 10, 14 in 16. Spojini 10 in 14 sta pokazali slabo selektivnost pri označevanju lipidnih kapljic. Spojini 7 in 16 sta bili učinkoviti pri označevanju plazemske membrane, pri čemer se je spojina 16 bolje vezala na membrano, medtem ko se je spojina 7 delno porazdelila tudi v notranje strukture celice. Spojina 7 se je izkazala za zelo fotostabilno in najbolj primerno za STED mikroskopijo, kar jo uvršča med obetavne kandidate za nadaljnje raziskave v superločljivostni mikroskopiji.

• Jezik: Slovenski jezik
• Ključne besede: STED, kumarini, fluorofori, lipidne kapljice, celična membrana
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2025
• PID: 20.500.12556/RUL-169895
• Datum objave v RUL: 14.06.2025

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Synthesis and characterization of fluorescent probes with a large Stokes shift for plasma membrane labelling

Izvleček

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.

• Ključne besede: STED, coumarins, fluorophores, lipid droplets, cell membrane