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V tem magistrskem delu so predstavljene metode za zaznavo treh elementov krvnega strdka – nanodelcev, fibrina in rdečih krvničk. Nanodelci so organski ali neorganski delci, ki imajo eno dimenzijo manjšo od 100 nm. Ker se je uporaba le-teh v zadnjem času močno razširila, obstaja vedno več predvsem epidemioloških študij, ki nanodelce povezujejo z različnimi obolenji, med drugim tudi s srčno-žilnimi boleznimi. Eno od bolezenskih stanj je nastanek krvnih strdkov. Krvni strdek je sestavljen iz fibrina, ki se polimerizira ob prisotnosti krvnih celic - trombocitov ter eritrocitov. Glavno vprašanje je, ali lahko vdihani nanodelci, ki so prisotni v onesnaženem zraku, povzročijo nastanek srčno-žilnih bolezni, oziroma natančneje, nastanek krvnega strdka. Problem pri preučevanju morebitne vzročne povezave med nanodelci in nastankom krvnih strdkov predstavlja detekcija vseh treh komponent v krvnem strdku ter detekcija nanodelcev, katerih velikost je pod resolucijo klasične fluorescenčne mikroskopije, zato smo želeli poiskati metodo, ki bi nam omogočila ločiti nanodelce, fibrin, eritrocite in trombocite v krvnem strdku. Metode, ki smo jih želeli preizkusiti za zaznavo so bile konfokalna fluorescenčna mikroskopija skupaj s fluorescenčno mikroskopijo s stimulirano emisijo in mikroskopsko slikanje življenjskega časa fluorescence. Vse metode temeljijo na pojavu fluorescence, zato smo uporabili fluorescentno označene nanocevke titanovega dioksida in fluorescentno označen fibrin, pri rdečih krvničkah smo zaznavali avtofluorescenco hemoglobina. Razvili smo metodo, s katero smo lahko ločili oba tipa celic (eritrocite in trombocite), fibrin ter nanodelce. Uporabljeno metodo bi bilo v prihodnje treba še nekoliko izpopolniti, da bi se lahko uporabljale ne le pri fiksiranih vzorcih, temveč tudi pri snemanju v realnem času. This master’s thesis presents the methods we wanted to use for detection three elements of a blood clot - nanoparticles, fibrin and, red blood cells. Nanoparticles are organic or inorganic particles measuring one dimension less than 100 nm. Since their use recently increased, there are more mainly epidemiological studies linking nanoparticles to a variety of diseases, including vascular. One of the disease states is the formation of a blood clot. A blood clot consists of fibrin, which polymerizes in the presence of blood cells - platelets and erythrocytes. The main question is whether inhaled nanoparticles present in polluted air can cause cardiovascular disease, or more specifically, the formation of a blood clot. The problem in studying possible causal link between nanoparticles and blood clot formation is the detection of all three components in a blood clot and the detection of nanoparticles whose size is below the resolution of classical fluorescence microscopy, so we wanted to find a method to separate nanoparticles, fibrin, erythrocytes and platelets in the blood clot. The methods we wanted to test for detection were confocal fluorescence microscopy along with stimulated emission depletion microscopy and fluorescence lifetime imaging microscopy. All of the methods are based on fluorescence, so fluorescently labeled titanium dioxide nanotubes and fluorescently labeled fibrin were used. In red blood cells, we detected auto-fluorescence of hemoglobin. We developed a method by which we were able to separate both cell types (erythrocytes and platelets), fibrin and nanoparticles. The method used should be further improved in the future so that they could be used not only for fixed samples but also in real-time experiments. |
