Real time optical coherence tomography monitoring of Candida albicans biofilm in vitro during photodynamic treatment

Abstract

The biofilm formed by Candida albicans is the mainly cause of infections associated to medical devices such as catheters. Studies have shown that photodynamic antimicrobial therapy (PAT) has lethal effect on <i>C. albicans</i>, and it is based on photosensitizer (PS) in the presence of low intensity light to generate reactive oxygen species in biological systems. The aim of this study was to analyze in real time, by Optical Coherence Tomography (OCT), the alterations in C. albicans biofilm in vitro during PAT using methylene blue (MB) as a PS and red light. An OCT system with working at 930nm was used, sequential images of 2000&times;512 pixels were generated at the frame rate of 2.5frames/sec. The dimension of the analyzed sample was 6000&mu;m wide by 1170&mu;m of depth corrected by refraction index of 1.35. We recorded 1min. before and after the irradiation with LED for PAT, generating 8min. of video. For biofilm formation, discs were made from elastomeric silicone catheters. The PS was dissolved in PBS solution, and a final concentration of 1mM MB was applied on biofilm, followed by a red LED irradiation (&lambda;=630nm&plusmn;20nm) during 6min. We performed a curve of survival fraction versus time of irradiation and it was reduced by 100% following 6min. of irradiation. OCT was performed for measurement of biofilm thickness of 110&mu;m when biofilm was formed. During irradiation, the variation of biofilm thickness was ~70&mu;m. We conclude that OCT system is able to show real time optical changes provided by PAT in yeasts organized in biofilm.