Sinteza fluorescentnih sond na osnovi barvil nilsko rdečega in nilsko modrega, primernih za fluorescenčno mikroskopijo s stimulirano emisijo

Stojanovski, Monika (author), Mravljak, Janez (mentor), Pajk, Stane (comentor)

Mikroskopija je znanost, katere začetki segajo daleč v 17. stoletje. Bila je prva, ki je omogočila opazovanje različnih majhnih objektov, ki s prostim očesom niso vidni. V osnovi se tehnika opazovanja ni veliko spreminjala, saj je bila velikost opazovanih objektov z uklonom svetlobe omejena na približno 200 nm. Pri večjih povečavah se svetlobni žarki uklonijo čez rob odprtine v mikroskopu in povzročijo zabrisano sliko opazovanega objekta. Govorimo o problemu ločljivosti optične mikroskopije, ki so ga znanstveniki v zadnjih desetletjih uspeli rešiti z razvojem super-ločljivostnih metod. Med te metode spada mikroskopija z vzbujenim praznjenjem emisije (STimulated Emission Depletion, STED), ki se v naravoslovnih vedah uporablja za opazovanje objektov nanometrskih velikosti. Pri tej metodi potrebujemo za selektivno opazovanje želenih objektov posebne fluorescentne sonde. Te fluorescentne sonde se razlikujejo po foto-fizikalnih lastnostih in po specifičnosti za vezavo na izbrane tarčne sestavine v vzorcu, zato jih je treba skrbno načrtovati glede na želeno uporabo. Primernih sond ni veliko, to pa prestavlja glavno oviro omenjene metode. V magistrski nalogi smo načrtovali in sintetizirali fluorescentne sonde za opazovanje lipofilnih celičnih struktur, ki bi bile primerne za STED. Za osnovo smo si izbrali nilsko rdeče in nilsko modro barvilo, ki se uporabljata v celični biologiji in histologiji. Pri nilsko rdečih derivatih smo osnovni benzofenoksazinski obroč modificirali tako, da se barvilo umešča v tekoče kristale in lipidne kapljice. Poskušali smo tudi s sintezo okoljsko občutljive membranske sonde na osnovi kumarinskega skeleta, ki bi omogočila razlikovanje med različno polarnimi območji celične membrane. Na žalost nam njena sinteza ni uspela. Dvema uspešno sintetiziranima sondama smo s spektrofotometrom in spektrofluorimetrom izmerili ekscitacijska in emisijska spektra, ki nakazujeta, da bi bili sondi primerni za STED mikroskopijo, kar so nato potrdili na Institutu Jožef Stefan. Ugotovili so, da sta se sondi odlično vmešali v tekoče kristale in sta primerni za STED mikroskopijo. V prihodnosti ju bodo preizkusili še na lipidnih kapljicah.

• Language: Slovenian
• Keywords: fluorescentne sonde, STED mikroskopija, nilsko rdeče barvilo, nilsko modro barvilo, kumarin
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2019
• PID: 20.500.12556/RUL-111158
• Publication date in RUL: 25.09.2019

Secondary language

• Language: English
• Title: Synthesis of fluorescent probes based on nile red and nile blue dyes, suitable for stimulated emission depletion microscopy

Abstract

Microscopy is a field of science which dates back to the 17th century. It was the first science to allow observation of various small objects that are invisible to the naked eye. The observation technique did not change much during history since the size of the observed objects was limited with the diffraction of the light (approximately to 200 nm). At larger magnification the light rays bend around the corners of the aperture inside the microscope causing a blurred image of the observed object. This is the so called problem of resolution in optical microscopy. Gratefully, the scientists were able to solve it with the development of super-resolution methods in the last few decades. One of this methods is called STimulated Emission Depletion (STED) microscopy and it's used to observe objects of nanometric scale. This method requires specifically designed fluorescent probes. These fluorescent probes differ according to photo physical properties and binding specificity for the selected target structures in the sample, that’s why we need to carefully design them in accordance with the desired use. This is the main obstacle of aforementioned method. During the research work for the thesis, we designed and synthesized lipophilic fluorescent probes suitable for STED microscopy of lipophilic cellular structures. Our probes are based on Nile red and Nile blue dyes which can be used in cellular biology and histology. We were able to modify their benzophenoxazine scaffolds in order to make the molecules able to interfere with liquid crystals and lipid droplets. We also tried to synthesize an environmental sensitive membrane probe, a derivative of coumarins. We wanted to make a probe which would make the differentiation between different polar areas of the cellular membrane possible. Unfortunately, we weren’t able to successfully produce the desired product. Two successfully synthesized probes expressed favourable photo physical properties and were sent to the Institute Jožef Stefan for further evaluation. They tested the use of our probes in STED microscopy. They concluded that both probes perfectly intertwined with liquid crystals. Furthermore, they are going to test the probes on liquid droplets as well.

• Keywords: fluorescent probes, STED microscopy, Nile red dye, Nile blue dye, coumarin