Candida albicans biofilm alterations during photodynamic therapy by optical coherence tomography video monitoring

Abstract

Reports in literature show that photodynamic therapy (PDT) presents a lethal effect on fungi, e.g. Candida albicans. It is based on the use of a photosensitizer (PS) in the presence of low intensity light to generate reactive oxygen species in biological systems. The purpose of this study was to analyze modifications in C. albicans biofilm in vitro during PDT using methylene blue (MB) and red light, with real time Optical Coherence Tomography (OCT). The OCT system used 930nm with 2mW of power and sequential images of 2000x512 pixels were generated at the frame rate of 2.5 frames/sec. The dimension of the analyzed sample was 6000 microns wide by 1170 microns of depth corrected by refraction index of 1.35. Images were recorded 1min before and 1min after the irradiation with LED for PDT, generating 1,200 images producing 8min of video. For biofilm growth, discs were made from elastomeric silicone catheters. The PS was dissolved in PBS solution, and a final concentration of 1mM MB was applied on the biofilm. A red LED (wavelenght = 630nm ± 20nm) was used for biofilm irradiation. 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, and it was 110 microns in the end time of biofilm formation. During the irradiation, the biofilm thickness changed in 70 microns. We conclude that OCT system is capable to show real time optical changes in yeasts organized in biofilm simultaneous to PDT.