The phenomenon of fluorescence was already used half a millennium ago for the purpose of determining the originality of the wood, based on the colouring of its extract. In the last two centuries, explanations of the phenomenon have been developed, new methods of use have been discovered and many fluorophores have been synthesized. Methods based on fluorescence have become invaluable in the fields of physics, biology, chemistry and medicine. And new techniques are continuously being introduced while the development of new fluorophores is lagging behind. Recently, a new fluorophore from polymethine family has been described in master's thesis of Andraž Bevk. Synthesis was subsequently optimized by Nataša Vek, described in detail in her master's thesis. In our master's thesis we investigated the nature of reactants that react under optimised conditions. Several different arylacetonitrils and ethyl arylacetates were tested. According to our results heteroaryl moiety must contain pyridine-like nitrogen, while the products of ethyl arylacetates undergo further cyclisation. Additionally, we determined the yields of three reactions with developed high performance liquid chromatography (HPLC) method.
Fluorophore emits photons after prior absorption. In addition to single-photon, the molecules are also capable of two-photon absorption. The theory of two-photon absorption was developed in the 1930s, but came into practice in the middle of the 20th century with the invention of strong enough lasers. Since the method of two-photon fluorescence microscopy has many advantages over the one-photon, the middle of the 20th century was also a time, when specialized two-photon probes were being first developed and studied. In the master's thesis we prepared a two-photon probe already described in the literature, and new coumarin-based two-photon probes. We encountered some problems during the synthesis of the latter and we were not able to prepare all the desired derivatives.
CNT were labelled with synthesized two-photon probes, however colleagues at Institut Jožef Stefan (IJS) were not able to detect any signal, probably because of the quenching of fluorescence by CNT.
Additionally, the excitation and emission spectra of synthesized fluorophores were recorded. Spectra of probe 1 are in accordance with the ones reported in the literature. Probe 7 stands out because of relatively large Stokes shift. And surprisingly, fluorophore 19 has significant blue-shift compared to the similar fluorophore 17.