Sinteza in vrednotenje sond na osnovi kumarinskega in fenoksazinskega skeleta za mikroskopijo z vzbujenim praznjenjem emisije

Gabrič, Alen (author), Mravljak, Janez (mentor), Pajk, Stane (comentor)

Mikroskopija z vzbujenim praznjenjem emisije (STED) je super-ločljivostna tehnika, ki omogoča preseganje difrakcijske limite, zaradi česar lahko natančneje preučujemo strukture, ki jih s klasičnimi mikroskopi ne moremo. Zaradi velike uporabnosti mikroskopov STED v naravoslovni znanosti, so tudi potrebe po specifičnih fluorescenčnih sondah velike. Za uporabo pri mikroskopiji STED morajo imeti sonde ustrezne ekscitacijske in emisijske spektre, ki sovpadajo z ekscitacijskim laserjem in laserjema STED, poleg tega pa ustrezno strukturo, ki omogoča selektivno označevanje le specifičnega mesta. V sklopu magistrske naloge smo sintetizirali dva tipa fluorescenčnih sond, primernih za preučevanje celičnih membran oz. tekočih kristalov in/ali lipidnih kapljic. Sonde temeljijo na kumarinskem in fenoksazinskem skeletu. Spojinam smo na koncu izmerili tudi ekscitacijske in emisijske spektre, na podlagi katerih smo potrdili vpliv določenih strukturnih fragmentov na ekscitacijo in emisijo. Končne spojine smo poslali na Institut Jožef Stefan, kjer so jih uporabili pri označevanju plazemske membrane oz. tekočih kristalov in/ali lipidnih kapljic, čemur je sledila mikroskopija STED. S sondami so označili biološke vzorce oz. tekoče kristale in lipidne kapljice ter ocenili selektivnost porazdeljevanja med celečimi membranami in označevanja tekočih lipidnih kapljic ter fotostabilnost in citotoksičnost. Vse štiri sintetizirane sonde so izpolnile pričakovanja glede selektivnosti označevanja. V prvem delu magistrske naloge smo primerjali sondi, ki sta bili sintetizirani z namenom označevanja plazemske membrane. Sonda, ki ima za osnovo diariloksazolni skelet, označuje predvsem celične membrane in nekoliko bolj fotobledi kot spojina, ki ima za osnovo kumarinski skelet. Kljub temu je lahko prva spojina dobro izhodišče za razvoj novih sond, ki imajo zelo velike Stokesove premike. Pri obeh sondah se lahko vidi tudi učinek uspešne uporabe dvojnega pozitivnega naboja, s čimer se spojini dlje časa zadržujeta v plazemski membrani. Ciano kumarin in derivat nilsko rdeče sta tudi bolj fotostabilna kot diariloksazolni derivat, kar je poledica kumarinskega skeleta, poleg tega pa v tem primeru signal prihaja predvsem iz lipidnih kapljic.

• Language: Slovenian
• Keywords: Mikroskopija z vzbujenim praznjenjem emisije / membranske sonde / dvojni naboj / kumarin / fenoksazin
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2019
• PID: 20.500.12556/RUL-109227
• Publication date in RUL: 28.08.2019

Secondary language

• Language: English
• Title: Synthesis and evaluation of probes for stimulated emission depletion microscopy based on coumarin and phenoxazine scaffolds

Abstract

Stimulated emission depletion (STED) microscopy is a super-resolution technique that allows the diffraction limit to be bypassed, which makes it possible to observe the structures that we can’t see with light microscopes. Due to high use of STED microscopes in natural science, the requirements for specific fluorescence probes are also high. For use in STED microscopy, the probes must have adequate excitation and emission spectra that coincide with excitation and STED lasers, and in addition, an appropriate structure that allows selective labeling of a specific area. Within the master’s thesis, two types of fluorescence probes suitable for the study of cell membranes and/ or liquid droplets and/or lipid droplets were synthesized. The probes are based on the coumarin and phenoxazine skeleton. In the end, we measured the excitation and emission spectra for the compounds based on witch we confirmed the influence of certain structural fragments on excitation and emission. The final compounds were sent to the Institute Jožef Stefan, where they were used in the labeling of the plasma membrane, liquid crystal and/or lipid drops, followed by the STED experiments. Biological samples and liquid crystals were labeled with the probes and evaluated based of the selectivity of distribution between membranes, selectivity of liquid lipid drops, photostability and cytotoxicity. All four synthesized probes met the expectations regarding selectivity of labeling. In the first part, we compared the probes that were synthesized in order to label the plasma membrane. The diaryloxazole skeleton based probe labels the cell membranes and photobleaches a little more than the coumarine based one. Nevertheless, the first compound may be a good starting point for the development of new probes that have very large Stokes shifts. For both probes, the effect of the successful use of a double positive charge can be seen, thus keeping the compounds for a longer time in the plasma membrane itself. The 4-cyano coumarin and the Nile red derivate are also more photostable than diaryloxazole derivative, which can be explained with the coumarine skeleton, moreover, the signal comes primarily from lipid droplets.

• Keywords: Stimulated emission depletion (STED) microscopy / membrane probes / double positive charge / coumarin / phenoxazine