Fluorescence is a form of photoluminescence, which was first documented as early as 16th and 17th century. A big milestone in its history represents the publication by George Gabriel Stokes in 1852, in which he also coined the term fluorescence. Nowadays fluorescence microscopy is an essential tool in biomedicine, biology and materials science, but was until recently restricted in its resolution by diffraction limit. That has changed with the invention of super resolution techniques, one of them being stimulated emission depletion microscopy (STED), which enable a resolution of a few tens of nanometers. Because super resolution techniques are relatively new, there is a shortage of commercially available fluorophores for certain applications. These also include labeling of biological membranes and carbon nanotubes, which represent a promising field of research.
Within the scope of this Master's thesis, we designed and synthesized coumarin and Nile Blue derivatives with various structural changes in order to adapt them to the greatest extent possible for labeling biological membranes and carbon nanotubues, improve photophysical properties and simultaneously make them suitable for stimulated emission depletion microscopy.
We successfully synthesized two Nile Blue and two coumarin derivatives. We evaluated individual synthesis steps and in the case of failure or lower yields determined plausible causes and suggested possible solutions. We assessed the impact of the planned structural changes on the photophysical properties of fluorophores and sent three compounds with favorable preliminary properties to further research at the Jožef Stefan Institute in Ljubljana. For the coumarin derivative 18, it was confirmed that it binds excellently on carbon nanotubes, but was also found that the brightness of the formed complex is inadequate, most likely because the tested type of carbon nanotubes quenches fluorescence. For compounds 8 and 11¸ it was determined, that they are unsuitable for use on mammalian cells, due to their nonspecific labeling of membranes, however the Nile Blue derivative 11 with the polar sulphonic head subsequently proved to be a good candidate for the visualization of the bacterial plasma membrane with stimulated emission depletion microscopy. Unfortunately, we did not achieve a significant improvement against photobleaching for any of the compounds, which represents a challenge for future research.
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