Depth penetration and detection of pH gradients in biofilms by two-photon excitation microscopy

Deep microbial biofilms are a major problem in many industrial, environment al, and medical settings. Novel approaches are needed to understand the str ucture and metabolism of these biofilms. Two-photon excitation microscopy ( TPE) and conventional confocal laser scanning microscopy (CLSM) were compar ed quantitatively for the ability to visualize bacteria within deep in vitr o biofilms. pH gradients within these biofilms were determined by fluoresce nce lifetime imaging, together with TPE. A constant depth film fermenter (C DFF) was inoculated for 8 h at 50 ml.h(-1) with a defined mixed culture of 10 species of bacteria grown in continuous culture. Biofilms of fixed depth s were developed in the CDFF for 10 or 11 days. The microbial compositions of the biofilms were determined by using viable counts on selective and non selective agar media; diverse mixed-culture biofilms developed, including a erobic, facultative, and anaerobic species. TPE was able to record images f our times deeper than CLSM. Importantly, in contrast to CLSM images, TPE im ages recorded deep within the biofilm showed no loss of contrast. The pH wi thin the biofilms was measured directly by means of fluorescence lifetime i maging; the fluorescence decay of carboxyfluorescein was correlated with bi ofilm pH and was used to construct a calibration curve. pH gradients were d etectable, in both the lateral and axial directions, in steady-state biofil ms. When biofilms were overlaid with 14 mM sucrose for 1 h, distinct pH gra dients developed. Microcolonies with pH values of below pH 3.0 were visible , in some cases adjacent to areas with a much higher pH (>5.0). TPE allowed resolution of images at significantly greater depths (as deep as 140 mu m) than were possible with CLSM. Fluorescence lifetime imaging allowed the in situ, real-time imaging of pH and the detection of sharp gradients of pH w ithin microbial biofilms.