Sinteza in vrednotenje fotofizikalnih lastnosti fluorescenčnih sond na osnovi 4-cianokumarina in nilsko rdečega za označevanje lipidnih kapljic

Krulec, Jona (author), Pajk, Stane (mentor)

Mikroskopija z vzbujenim praznjenjem emisije (STED) spada med superločljivostne tehnike in predstavlja eno izmed najuporabnejših tehnik za opazovanje nanometrskih struktur v celicah. Tehnika temelji na fluorescenci, fizikalnem pojavu pri katerem snov najprej absorbira elektromagnetno valovanje, čemur nato sledi emisija elektromagnetnega valovanja pri višji valovnih dolžinah. Kakor pri večini fluorescenčnih mikroskopij, tudi pri STED mikroskopiji uporabljamo fluorofore, s katerimi označimo opazovano strukturo. Lastnosti teh se sicer razlikujejo, predvsem morajo imeti dobro fotostabilnost ter ustrezen emisijski in absorpcijski spekter. Ker želimo, da se fluorofori umeščajo le v določene strukture oz. dele celičnih struktur, je pomembna njihova selektivnost. Namen našega dela je bila sinteza novih fluorescentnih sond in preverjanje ustreznosti sintetiziranih sond za označevanje celičnih struktur na STED mikroskopu. Izhodišče magistrske naloge so predstavljale sonde za označevanje lipidnih kapljic na osnovi 4-cianokumarina in nilsko rdečega, ki so bile pripravljene v predhodnih magistrskih nalogah. Oba tipa sond sta se izkazala kot zelo ustrezna za STED mikroskopijo, vendar so se pojavile težave s selektivnostjo, saj je kar nekaj signala prihajalo iz ozadja. Spojine, ki smo jih sintetizirali (spojini 18 in 20), imajo nekoliko večjo lipofilnost, zato smo pričakovali večjo selektivnost pri označevanju lipidnih kapljic. Poleg sond za označevanje lipidnih kapljic smo pripravili tudi sondo za označevanje plazemske membrane (spojina 11) na osnovi nilsko rdečega. Emisija nilsko rdečega je zelo občutljiva na polarnost okolice, zato se nadejamo, da bo sonda uporabna pri spremljanju procesov na membrani, ki vključujejo spremembe v polarnosti. Sondam 11, 18 in 20 smo pomerili absorpcijske in emisijske spektre in ti ustrezajo specifikacijam, ki jih zahteva STED mikroskop na Institutu Jožef Stefan. Sintetizirane sonde so testirali kolegi na STED mikroskopu na Institutu Jožef Stefan. Ugotovili so, da sondi 18 in 20 selektivno označujeta lipidne kapljice, nista citotoksični, težavo pa predstavlja fotoblednje obeh sond. Slednje nas je presenetilo, saj sonde s popolnoma enakim fluoroforom, ki so bile osnova za naše sonde, niso bile tako zelo dovzetne za razpad na svetlobi. Testi s sondo 11 bodo opravljeni v prihodnosti.

• Language: Slovenian
• Keywords: STED, nilsko rdeče, 4-cianokumarin, lipidne kapljice, fluorescenčne sonde
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2020
• PID: 20.500.12556/RUL-115960
• Publication date in RUL: 03.05.2020

Secondary language

• Language: English
• Title: Synthesis and evaluation of photophysical properties of 4-cyanocumarin- and nile red-based fluorescent probes for labelling lipid droplets

Abstract

Stimulated emission depletion microscopy is one of the super-resolution techniques as well as one of the most useful techniques for observing nanometer structures in cells. It is based on the use of fluorescence; the absorption of light, followed by the emission of light at higher wavelengths. As with most fluorescence microscopes, STED microscopy uses fluorophores to label the observed structure. The properties of these fluorophores differ; in particular they must have better photostability as well as an appropriate emission and absorption spectrum. Since we want the fluorescent labels to distribute to certain structures or parts of structures, their selectivity is very important. The purpose of our work was synthesis of new fluorescent probes and checking their suitability for marking cell structures on a STED microscope. The basis for my master's thesis were probes for the labelling of lipid droplets based on 4-cyanocoumarin and Nile red, which have already been introduced in various other master's thesis. Both types of probes proved to be very suitable for STED microscopy, but there were problems with selectivity as there was a lot of signal from the background. The compounds described in this master's thesis (compounds 18 and 20), however, have a slightly higher lipophilicity, so we expected greater selectivity in the labelling of lipid droplets. In addition to lipid droplet labelling probes, a plasma membrane labelling probe (compound 11) based on Nile red was also prepared. The Nile red emission is very sensitive to the polarity of the surroundings, so we hope that the probe will be useful in monitoring membrane processes involving changes in polarity. The absorption and emission spectra were recorded for compounds 11, 18 and 20, and these in principle correspond to the specifications required by the STED microscope at the Jožef Stefan Institute. Tests done at institut have shown that probes 18 and 20 more selectively label lipid droplets, are not cytotoxic, but photobleaching of both probes is a problem. This surprised us, because probes that have exactly the same fluorophore and were the basis for our probes, do not photobleech as much. Tests on STED microscope with probe 11 will be done in the future.

• Keywords: STED, Nile red, 4-cyanocoumarin, lipid droplets, fluorescent probes