| dc.description | Antimicrobial photodynamic therapy (APDT) is a promising tool to counterattack the emerging
treat of drug-resistant pathogens. The technique combines low-intensity monochromatic light
with a photosensitizer compound to produce reactive oxygen species (ROS) that can damage
virtually any type of biomolecules and lead to rapid ce\l death. Since some ROS present
diffusion-limited reactivity, most cell damage is co-localized with photosensitizer accumulation
site. Hence, imaging photosensitizer accumulation and fluorescence lifetime in the nanoscale
can bring a great levei of information to further understand the ultrastructural cellular damage
caused by APDT. In this study, we used a FLIM system capable of single-molecule detection to
observe the accumulation and interaction sites of methylene blue (MB), a very broadly-used
photosensitizer, in yeast, and Gram-positive and Gram-negative bacterial cells, Our data shows
fluorescence lifetime contrast, with nanometric resolution, among different cellular structures
such as cell wall, membrane and DNA. The images evidentiate differential MB accumulation in
microbial cells and the existence of two different populations of MB molecular species: those
interacting mostly with the solvent (short-lived, - 0.8 ns) and those interacting with
biomolecules (Iong-lived, -2 ns), The short-lived fluorescence predominates in the mucoid
capsule of Gram-negative bacteria and cell-wall ofyeast and Gram-positive bacteria while longlived
MB fluorescence shows preferential accumulation in DNA-rich sites 1
??? It is marked in yeast
nucleus and exclusively inside bacterial cells. In fact, literature supports that MB intercalation in
nucleic acids stabilizes its excited-states leading to increased "fluorescence "lifetime and
efficiency of singlet-oxygen production2
. Our data brings evidence that this sOli of phenomena
can be observed by FLIM in the nanoscale and this should bring new insights to the
photophysical, photochemical and biological mechanisms of photodynamic therapy. | |
