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17 May 2010Assessment of photodynamic damage on Escherichia coli via atomic force microscopy
1Instituto de Pesquisa em Saúde (Brazil) 2Instituto de Pesquisas Energéticas e Nucleares (Brazil) 3São Leopoldo Mandic (Brazil) 4Instituto de Estudos Avançados (Brazil)
Photodynamic antimicrobial therapy (PAT) may become a useful clinical tool to treat microbial infections, overcoming
microbial resistance that is a major problem nowadays. The aim of our work was to verify the damage caused by
photosensitization over a Escherichia col) via atomic force microscopy (AFM), looking for structural changes that might
occur in cells after PAT. Cells culture were grown until a stationary phase to reach a concentration of approximately 108
cells/mL allowing the production of extracellular slime in a
biofilm-like structure. The cells including the extracellular
matrix were put in a slide and its structure was observed using AFM; subsequently a water solution of methylene blue at
60μM was applied over the cells and a pre-irradiation time of 3 minutes was waited and followed by illumination with a
diode laser (λ=660nm, power 40mW, 3min, fluence 180J/cm2, beam diameter 0.04cm2). The same cells were observed
and the images stored. A second set of experiments was performed with a smaller number of cells/area and without
extracellular slime, using the parameters abovementioned. The results showed alterations on cellular scaffold markedly
dependent on the number of cells and the presence of extracellular slime. The slime is targeted by the photosensitizer,
and after irradiation a destruction of the matrix was observed; when fewer cells were evaluated the destruction is much
more evident. The images suggested rupture of the cellular membrane and cellular fragments were observed. Our
findings indicate that AFM seems is a useful tool to investigate parameters linked with photodestruction of
microorganisms.
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Silvia Cristina Núñez, Martha Simões Ribeiro, Aguinaldo Silva Garcez, Walter Miyakawa, "Assessment of photodynamic damage on Escherichia coli via atomic force microscopy," Proc. SPIE 7715, Biophotonics: Photonic Solutions for Better Health Care II, 77151L (17 May 2010); https://doi.org/10.1117/12.854589