Sinteza in vrednotenje fluoroforov s fenoksazinskimi in kumarinskimi skeleti, primernih za fluorescenčno mikroskopijo membran s stimulirano emisijo

Novak, Matej (author), Pajk, Stane (mentor)

STED nanoskopija je super-ločljivostna mikroskopska metoda, ki se uporablja v naravoslovnih znanostih za opazovanje struktur, ki so manjše od difrakcijske omejitve svetlobe, kar je glavna prednost glede na klasičen fluorescentni mikroskop. Predpogoj za STED pa so primerne fluorescenčne sonde, ki omogočajo metodi selektivno vklapljanje in izklapljanje fluorescence, ki v tem trenutku predstavljajo največjo oviro tej metodi. V magistrski nalogi smo načrtovali in sintetizirali fluorescenčne sonde, ki bi omogočale selektivno označevanje celičnih membran. Sonde temeljijo na fenoksazinskem in kumarinskem skeletu, ki pripadata že znanim fluoroforom. Številni fluorofori na podlagi omenjenih skeletov še niso bili preizkušeni pri STED nanoskopiji. Na osnovne skelete smo pripeli funkcionalne skupine, ki omogočijo sondam umeščanje v membrane. V okviru te naloge smo preizkusili in optimizirali reakcije in sintetizirali večje število potencialnih sond. Vsem smo izmerili ekscitacijske in emisijske spektre, na podlagi katerih smo povezali vpliv strukture na foto-fizikalne lastnosti. Izbrane sonde, to so PNMb-8, PNMb-9, PNMaw-2, PNMbw-2, PNMkb-2, PNMpb-2 in PNMpa-2, smo poslali na Institut Jožef Stefan, kjer so jih uporabili v preizkusu na STED. S sondami so označili biološke vzorce in jih ocenili glede na selektivnost porazdeljevanja med celičnimi membranami, emisijo pod pogoji STEDa, fotostabilnostjo in citotoksičnostjo. Sintetizirane sonde dobro označijo plazemsko membrano, vendar hitro poteče internalizacija do drugih celičnih membran. Najbolj je izstopala sonda PNMpa-2, ki omogoča pridobitev odličnih super-ločljivih slik, ni citotoksična in je zelo fotostabilna, tudi v primerjavi s komercialno dostopnimi sondami. Sonda temelji na kumarinskem skeletu z N-metilpiridinsko skupino na mestu 3 in predstavlja dobro izhodišče za nadaljnji razvoj primernih sond. Največja pomanjkljivost fenoksazinskih sond, kot je PNMbw-2, je omejena fotostabilnost in bi jih bilo smiselno razvijati v smeri izboljšanja na tem področju. Kljub temu, da sonda PNMbw-2 zaradi fotobledenja ni primerna za večkratno zajemanje slike, so le te izjemne ločljivosti. Sonda PNMb-9 pa je izkazovala citotoksičnost kljub zelo ostrim STED slikam in zanemarljivemu fotobledenju. Ali je to značilnost vseh 4-cianokumarinov, bi morali potrditi s testiranjem dodatnih sond.

• Language: Slovenian
• Keywords: membranske sonde, STED, super-ločljivost, kumarin, fenoksazin
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2019
• PID: 20.500.12556/RUL-106849
• Publication date in RUL: 21.03.2019

Secondary language

• Language: English
• Title: Synthesis and evaluation of fluorophores with phenoxazine and coumarin scaffolds applicable for stimulated emission depletion microscopy of membranes

Abstract

STED nanoscopy is a type of super-resolution microscopy used to observe structures smaller than the diffraction limit of light, which is also its main advantage compared to classic fluorescent microscopy. In order to function, STED requires compatible fluorescent probes which are able to be turned on or off on demand. These probes are momentarily the biggest obstacle for STED. During this work, we designed and synthesized fluorescent probes able to selectively stain cell membranes. They are based on phenoxazine and coumarin scaffolds which are already present as known fluorophores, yet many variations have not been tested in STED nanoscopy. The basic scaffolds were further modified with various functional groups to enable the probes to associate with membranes. We optimised the reactions and synthesized an array of probe candidates and measured their excitation and emission spectra that were used as basis to evaluate the influence of structure on photo-physical attributes. The chosen probes, which are PNMb-8, PNMb-9, PNMaw-2, PNMbw-2, PNMkb-2, PNMpb-2 and PNMpa-2, were sent to Institute Jožef Stefan and used in STED experiments. They were used to stain biological samples and evaluated in the following criteria: selectivity of distribution between cell membranes, emission under STED conditions, photostability and cytotoxicity. Our probes label the plasma membrane very quickly, but they also stain other membranes rapidly. The best results were provided by PNMpa-2 that gives excellent super-resolution images, is not cytotoxic and is very photostable, even compared to commercially available probes. It is based on the coumarin scaffold with a N-methylpyridine group on position 3 and is a good starting point for further development of similar probes. The biggest drawback of phenoxazine probes, like PNMbw-2, is their limited photostability, which should be focused on first, during further development. Images taken with PNMbw-2, have amazing resolution, yet the probe was only suitable for a couple of pictures. The probe PNMb-9 showed signs of cytotoxicity, but on the other hand provided very sharp STED images and photobleached very lightly. Whether this is a trait of all 4-cyanocoumarins, has yet to be proven with further testing of additional probes.

• Keywords: membrane probes, STED, super-resolution, coumarin, phenoxazine