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

Abstract

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.

Keywords: Candida albicans; LED illumination; antimicrobial photodynamic therapy; fluorescence microscopy; hypericin; image analysis; kinetics; photosensitizer uptake.

Figure 1

The structure of Hypericin.

Figure 2

Time series of fluorescence micrographs illustrating hypericin uptake in yeast cells at 1, 3, 5, 7, 10, 15, 20, 25, 30, 35, 40, and 45 min. Images correspond to the expanded second stage of the experiment and were used for quantitative ImageJ analysis of fluorescence area (%Area) following a fixed threshold based on the control sample (Magnification ×600).

Figure 3

Kinetics of hypericin fluorescence in Candida albicans under in vitro conditions.

Figure 4

Kinetics of hypericin fluorescence in Candida albicans under dark conditions.

Figure 5

Representative dark-series micrographs illustrating minimal and subtle fluorescence signals in yeast cells in the absence of light activation. Owing to the low contrast between background and cellular fluorescence, visual differentiation was limited, and quantitative evaluation required threshold-based image analysis using specialized software (Magnification ×600).