Molekulární diagnostika pomocí nanočástic s použitím metod časově rozlišené fluorescenční mikroskopie

Abstract

Casove rozlisena uorescencn mikroskopie je opticka metoda, ktera umoznuje zobrazen a analyzu molekularnch procesu v prirozenem biologickem prostred. Lze ji uplatnit naprklad pri detekci uorescencnch castic, stanoven vlastnost bezprostrednho okol uoroforu ci sledovan interakce nanocastic s jinymi latkami. V prvn casti teto bakal arske prace je uvedena teorie k problematice se zamerenm na princip uorescence, urcovan doby zivota a nanocastice (zejmena diamantove). Ve druhe casti jsou popsany rozdly mezi casove rozlisenou a nerozlisenou metodou, optimalizacn postupy prpravy vzorku nanodiamantovych castic a provedene experimenty. Vysledkem optimalizace jsou predevsm nalezen vhodne koncentrace roztoku nanodiamantu a jejich uprava pomoc kationickeho polymeru. Hlavnm vysledkem provedenych experimentu je vytvoren metody, kterou lze detekovat navazan polymeru na diamantove nanocastice. Tyto castice byly pote dopraveny do bunek.

Fluorescence lifetime imaging is an optical method which is used for imaging and analysis of molecular processes in biological environment. One of the possible uses is detection of uorescent particles, determination of properties in surrounding uorophore or observation of nanoparticles' interaction with other materials. In the rst part of this bachelor's work, there is described a theory regarding principles of uorescence and nanoparticles (especially nanodiamonds). There are also comparison of time resolved andstandard confocal methods, optimalizations of procedures of sample preparation and experiments in the second part. The results of optimalization procedures are mainly a suitable solution concentration and its adjustment with cationic polymer. The main result is a method, which allows to detect binding of polymer to nanoparticles. These nanoparticles were then transported into cells.