Abstract
Antibacterial treatments based on photosensitized production of reactive oxygen species is a promising approach to address local microbial infections. Given the small size of bacterial cells, identification of the sites of binding of the photosensitizing molecules is a difficult issue to address with conventional microscopy. We show that the excited state properties of the naturally occurring photosensitizer hypericin can be exploited to perform STED microscopy on bacteria incubated with the complex between hypericin and apomyoglobin, a self-assembled nanostructure that confers very good bioavailability to the photosensitizer. Hypericin fluorescence is mostly localized at the bacterial wall, and accumulates at the polar regions of the cell and at sites of cell wall growth. While these features are shared by Gram-negative and Gram-positive bacteria, only the latter are effectively photoinactivated by light exposure.
Highlights
We have recently shown that the protein apomyoglobin can be usefully exploited as a biocompatible nanocarrier for Hyp since it preserves its fluorescence, photodynamic properties and bacterial photoinactivation ability[3,26,27]
Not all fluorophores are amenable to STimulated Emission Depletion (STED) superresolution microscopy, especially if excited state absorption bands are present in the spectral region where excited depletion is performed[28,29]
Subtracting the ground state absorption from the transient absorption spectrum allows removing the contribution of ground state bleaching, as exemplified in Fig. 1B for the data collected at 100 ps delay
Summary
We have explored the possibility to exploit the intense fluorescence emission of a naturally occurring PS molecule, hypericin (Hyp), to identify its distribution inside living bacteria using a STimulated Emission Depletion (STED) microscopy approach. Results Evidence of stimulated emission by Hyp-apoMb from femtosecond pump-and-probe transient absorbance.
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