Fluorescence microscopy is a sub-type of optical microscopy that uses fluorescence or
phosphorescence to study a sample. It is an indispensable method in cell biology, where specific
organelles or molecules are labeled with fluorescent dyes, whose fluorescence is efficiently
separated from the background signal due to specific emission. These dyes are called
fluorophores. They are molecules that absorb light at a certain wavelength and are excited to a
higher energy state, emitting a photon with a lower wavelength when they return to their ground
state. Their best-known application is in the labeling of cell organelles. When selecting a suitable
fluorophore for fluorescence microscopy, some of its key properties, such as absorption and
emission spectra, molar extinction coefficient, quantum yield, efficiency and selectivity of
labeling of cell organelles, chemical stability and photostability, have to be taken into account.
The major drawback of conventional fluorescence microscopy is poor resolution, which has been
successfully addressed by the development of super-resolution methods. One of them is called
stimulated emission depletion (STED) microscopy, which was first mentioned in 1994. This is
mostly used in the life sciences to observe objects smaller than the diffraction limit (< 200 nm).
For the purpose of this thesis, 5 fluorescent probes synthesised at the Faculty of Pharmacy,
University of Ljubljana were analysed. In the first part, which took place at the Faculty of
Pharmacy, the photophysical properties of the fluorescent probes were evaluated. The absorption
(excitation) and emission spectra were measured on a UV-VIS and a fluorescence spectrometer,
and the molar extinction coefficient and quantum yield were calculated. In the second part, we
then determined the suitability of each fluorescent probe for labeling lipid droplets of LA-4 cells
by confocal fluorescence microscopy and STED microscopy. We were interested in whether the
probe is suitable for STED microscopy, which cell organelles it labels, its photostability and
potential cytotoxicity. This part of the work was carried out at the Jožef Stefan Institute in
Ljubljana.
We found that the probes were not the most suitable for STED microscopy, because they gave
images with only slightly improved resolution. Some probes labeled lipid droplets and were
spectrally sensitive, while others labeled other internal cellular structures. One probe had
cytotoxic properties. All of them were extremely photostable, which could be used in
combination with other probes, suitable for STED microscopy.
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