20.500.12556/RUL-159252 Vrednotenje fotofizikalnih lastnosti izbranih fluorescenčnih sond in njihove primernosti za označevanje lipidnih kapljic Evaluation of selected fluorescent probes’ photophysical properties and their suitability for lipid droplets labeling Fluorescenčna mikroskopija je ena od podvrst optične mikroskopije, kjer za proučevanje vzorca uporabljamo fluorescenco ali fosforescenco. Gre za nepogrešljivo metodo v celični biologiji, pri kateri specifične organele oz. molekule označimo s fluorescenčnimi barvili, katerih fluorescenco zaradi specifične emisije učinkovito ločimo od signala ozadja. Ta barvila imenujemo fluorofori. To so molekule, ki pri določeni valovni dolžini absorbirajo svetlobo ter preidejo v višje energijsko stanje, ko se vrnejo v osnovno stanje, pa oddajo foton z daljšo valovno dolžino. Najbolj poznano področje njihove uporabe je označevanje celičnih organelov. Pri izbiri primernega fluorofora za fluorescenčno mikroskopijo moramo upoštevati nekatere njegove ključne lastnosti, kot so ekscitacijski in emisijski spekter, molarni ekstinkcijski koeficient, kvantni izkoristek, učinkovitost in selektivnost označevanja želenih celičnih struktur, kemijska stabilnost in fotostabilnost. Pomanjkljivost klasične fluorescenčne mikroskopije je slaba ločljivost, ki pa so jo znanstveniki uspešno rešili z razvojem super-ločljivostnih metod. Ena izmed le-teh je mikroskopija z vzbujenim praznjenjem emisije (STED, angl. stimulated emission depletion), ki je bila prvič omenjena leta 1994. Največkrat jo uporabljamo za proučevanje objektov, manjših od uklonske limite (< 200 nm). Za namen te magistrske naloge smo analizirali 5 na Fakulteti za farmacijo, Univerze v Ljubljani, sintetiziranih fluorescenčnih sond. V prvem delu, ki je potekal na Fakulteti za farmacijo, smo vrednotili fotofizikalne lastnosti fluorescenčnih sond. Na UV-VIS ter fluorescenčnem spektrometru smo jim pomerili absorpcijski (ekscitacijski) ter emisijski spekter ter izračunali molarni ekstinkcijski koeficient in kvantni izkoristek. V drugem delu smo nato ugotavljali primernost posamezne fluorescenčne sonde za označevanje lipidnih kapljic celic LA-4 z metodama konfokalne fluorescenčne mikroskopije in STED mikroskopije. Zanimalo nas je, ali je sonda primerna za STED mikroskopijo, katere celične organele še označuje, kakšna je njena fotostabilnost in potencialna citotoksičnost. Ta del je potekal na Institutu Jožef Stefan v Ljubljani. Ugotovili smo, da sonde niso najbolj primerne za STED mikroskopijo, saj smo z njo pridobili posnetke z zgolj malo izboljšano ločljivostjo. Nekatere sonde so označile lipidne kapljice in so bile spektralno občutljive, nekatere pa so označile druge notranje celične strukture. Ena sonda je imela citotoksične lastnosti. Vse so bile izjemno fotostabilne, zaradi česar bi jih lahko uporabili v kombinaciji z drugimi sondami, ki pa so primerne za STED mikroskopijo. Fluorescence microscopy is a sub-type of optical microscopy that uses fluorescence or phosphorescence to study a sample. It is an indispensable method in cell biology, where specific organelles or molecules are labeled with fluorescent dyes, whose fluorescence is efficiently separated from the background signal due to specific emission. These dyes are called fluorophores. They are molecules that absorb light at a certain wavelength and are excited to a higher energy state, emitting a photon with a lower wavelength when they return to their ground state. Their best-known application is in the labeling of cell organelles. When selecting a suitable fluorophore for fluorescence microscopy, some of its key properties, such as absorption and emission spectra, molar extinction coefficient, quantum yield, efficiency and selectivity of labeling of cell organelles, chemical stability and photostability, have to be taken into account. The major drawback of conventional fluorescence microscopy is poor resolution, which has been successfully addressed by the development of super-resolution methods. One of them is called stimulated emission depletion (STED) microscopy, which was first mentioned in 1994. This is mostly used in the life sciences to observe objects smaller than the diffraction limit (< 200 nm). For the purpose of this thesis, 5 fluorescent probes synthesised at the Faculty of Pharmacy, University of Ljubljana were analysed. In the first part, which took place at the Faculty of Pharmacy, the photophysical properties of the fluorescent probes were evaluated. The absorption (excitation) and emission spectra were measured on a UV-VIS and a fluorescence spectrometer, and the molar extinction coefficient and quantum yield were calculated. In the second part, we then determined the suitability of each fluorescent probe for labeling lipid droplets of LA-4 cells by confocal fluorescence microscopy and STED microscopy. We were interested in whether the probe is suitable for STED microscopy, which cell organelles it labels, its photostability and potential cytotoxicity. This part of the work was carried out at the Jožef Stefan Institute in Ljubljana. We found that the probes were not the most suitable for STED microscopy, because they gave images with only slightly improved resolution. Some probes labeled lipid droplets and were spectrally sensitive, while others labeled other internal cellular structures. One probe had cytotoxic properties. All of them were extremely photostable, which could be used in combination with other probes, suitable for STED microscopy. fluorescenčne sonde molarni ekstinkcijski koeficient kvantni izkoristek STED mikroskopija lipidne kapljice fluorescent probes molar extinction coefficient quantum yield STED microscopy lipid droplets true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2024-07-04 08:45:15 2024-07-04 08:45:19 2024-07-07 03:19:09 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 105151 21068.pdf 21068.pdf 1 FB139BCA07987E1E291A0916B2865BAD 1700975faba15cfd723b4937b5e3ef48e3dcd5a6ae39c6c86a351508f4241b74 fa0ecb46-39d0-11ef-90ba-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=187209 Fakulteta za farmacijo 0 0 0