20.500.12556/RUL-158973 Sinteza in vrednotenje fluorescenčnih označevalcev lipidnih kapljic, primernih za mikroskopijo z vzbujenim praznjenjem emisije Synthesis and evaluation of fluorescent labels for lipid droplets suitable for stimulated emission depletion microscopy Fluorescenca je vrsta luminiscence, pri kateri fluorofor absorbira svetlobo določene valovne dolžine, kar povzroči, da fluorofor preide v vzbujeno stanje. Po določenem času (nekaj nanosekund), fluorofor emitira foton in spontano preide nazaj v osnovno stanje. Ta fenomen izkoriščamo pri fluorescenčni mikroskopiji, ki je nepogrešljivo orodje pri praktično vseh bioznanostih. Nadgradnja fluorescenčne mikroskopije je mikroskopija z vzbujenim praznjenjem emisije (STED), saj lahko z njo opazujemo veliko manjše strukture nanometrskih velikosti. Fluorofori, ki se uporabljajo za super resolucijsko tehniko STED, imajo posebne lastnosti, saj morajo biti fotostabilni, imeti morajo ustrezni absorpcijski in emisijski spekter, ter specifično označit strukture, ki jih želimo opazovati. Namen magistrske naloge je bil razvoj sinteze novih merocianinov (derivatov kumarina) z namenom, da bi jih s strukturnimi spremembami pretvorili v dobre označevalce lipidnih kapljic, ter izboljšali lastnosti za mikroskopijo z vzbujenim praznjenjem emisije (STED). Nekaj tovrstnih označevalcev so že sintetizirali na Fakulteti za farmacijo, vendar so bili izkoristki nizki, tudi zaradi nastanka stranskih produktov. Poleg tega smo želeli raziskati kako različne elektron-privlačne in elektron-donorske skupine vplivajo na fotofizikalne lastnosti načrtovanih sond. Zato smo se lotili sintezne poti drugače in pripravili 8 novih fluorescenčnih sond, katerim smo pomerili ekscitacijski in emisijski spekter. 5 pripravljenih sond (7, 9, 11, 12 in 14) je imelo ustrezne spektralne lastnosti, da smo z njimi nadaljevali eksperimente na STED mikroskopu na Inštitutu Jožefa Stefana (IJS). S pripravljenimi sondami smo inkubirali celice celične linije LA-4. Glede na lastnosti označevanja podobnih sond, smo pričakovali, da bodo naše sonde označile lipidne kapljice. To so relativno neraziskani celični organeli, ki pa so nedvomno vpleteni v razvoj določenih rakavih in infekcijskih bolezni. Po eksperimentih na STED mikroskopu smo ugotovili, da pripravljene sonde niso izkazovale citotoksičnosti, po fotostabilnosti pa je izstopala sonda 11. Vendar pa nobena izmed sond ni zadovoljivo označevala lipidnih kapljic, ker so sonde nespecifično označevale druge organele znotraj celice. 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. STED merocianini lipidne kapljice kumarini fluorescenca STED merocyanines lipid droplets coumarins fluorescence true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2024-06-26 07:45:14 2024-06-26 07:45:30 2024-07-07 03:24:31 0000-00-00 00:00:00 2024 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 105091 21084.pdf 21084.pdf 1 B8BCCCBCA8CDDE96D552AAF0A38FA353 01efb2e30318335b3a89ea26a470b526b8d7949618a6b5fe4d124234fcd0b153 53635b0d-337f-11ef-8fa5-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=186712 Fakulteta za farmacijo 0 0 0