Analysis of the Uptake of Hypericin by Candida albicans Yeast Cells Using Fluorescence Methods and Comparison of the Dynamics of This Process over Time.

Background: Hypericin is a natural photosensitizer with promising antifungal potential, but its uptake kinetics in Candida (C.) albicans are not well defined. Objective: To characterize the time-dependent uptake of hypericin by C. albicans in vitro using fluorescence microscopy and quantitative image analysis. Methods: C. albicans ATCC 90028 was standardized to 0.5 McFarland and incubated with hypericin dissolved in DMSO. Samples were illuminated with an LED system tuned near 550 nm and imaged using a CCD fluorescence microscope with emission recorded above ≈600 nm. Images were analyzed in ImageJ, using a control-based threshold and percentage area (the percentage of pixels above the threshold in the whole field) as a fluorescence measure. Time points were 1, 3, 5, 7, 10, 15, 20, 25, 30, 35, 40, and 45 min, plus a separate dark-only series at 35-45 min. Data from three experiments were evaluated by ANOVA. Results: Fluorescence increased rapidly and showed a nonlinear, biphasic profile under light, with local maxima around 5-7 and 15-30 min. Dark-only samples at 35-45 min had a lower %Area and lacked a clear biphasic pattern. Conclusions: Hypericin uptake by C. albicans is dynamic, nonlinear, and light-dependent. These kinetics should be considered when designing hypericin-based antifungal photodynamic therapy protocols.