Biofilms are the most widespread form of bacterial growth in nature, as they provide protection against environmental stress factors. As bacteria in biofilms are more resistant to antimicrobial agents, it is more difficult to remove them and to disinfect the surfaces on which they grow. When biofilms are formed by bacteria that are pathogenic to animals and humans, they also pose a serious threat to their health. The structure of biofilms can be observed using fluorescence microscopy, but this requires the use of suitable fluorescence probes. There are very few commercially available probes, and the structural elements that would allow the probes to bind to elements of the bacterial film are not well known.
As part of the master's thesis, we therefore synthesised several fluorescent probes to which we bound different azides to Nile red by click chemistry, varying their lipophilicity, polarity, and charge. Nile red was chosen because it is an environmentally sensitive fluorophore that emits very poorly in aqueous (polar) environments. We therefore assumed that emission would only occur after binding to biofilm structures.
We successfully synthesised six derivatives of Nile red and measured the excitation and emission spectra for selected compounds. All six compounds were then tested at the Jožef Stefan Institute in Ljubljana, by assessing the labeling of the bacterial biofilm with the newly synthesised probes was verified using model biofilms of Listeria monocytogenes and Salmonella enterica serovar Infantis and a confocal fluorescence microscope. At the same time, we also checked whether the probes caused changes in the biofilm structure or inhibited bacterial growth. We found that the synthesised probes uniformly labelled the bacterial population in the biofilms of L. monocytogenes, whereas in S. enterica only some of the bacteria were labelled, which could be attributed to differences in the composition of the model biofilms.
The prepared probes were also used as selective markers for eukaryotic cell organelles and analysed using a stimulated emission discharge (STED) microscope, a fluorescence microscopy technique that allows imaging with higher spatial resolution than that given by the diffraction limit of light. All probes were suitable for STED microscopy, as we observed very good image resolution. However, their signal faded moderately, making them unsuitable for experiments with prolonged exposure to excitation and STED laser. Nevertheless, the selected prepared fluorescent probes effectively labelled mitochondria and/or lysosomes.
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