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

Abstract

Deep microbial biofilms are a major problem in many industrial, environmental, and medical settings. Novel approaches are needed to understand the structure and metabolism of these biofilms. Two-photon excitation microscopy (TPE) and conventional confocal laser scanning microscopy (CLSM) were compared quantitatively for the ability to visualize bacteria within deep in vitro biofilms. pH gradients within these biofilms were determined by fluorescence lifetime imaging, together with TPE. A constant-depth film fermentor (CDFF) 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 depths 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 nonselective agar media; diverse mixed-culture biofilms developed, including aerobic, facultative, and anaerobic species. TPE was able to record images four times deeper than CLSM. Importantly, in contrast to CLSM images, TPE images recorded deep within the biofilm showed no loss of contrast. The pH within the biofilms was measured directly by means of fluorescence lifetime imaging; the fluorescence decay of carboxyfluorescein was correlated with biofilm pH and was used to construct a calibration curve. pH gradients were detectable, in both the lateral and axial directions, in steady-state biofilms. When biofilms were overlaid with 14 mM sucrose for 1 h, distinct pH gradients 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 microm) 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 within microbial biofilms.

FIG. 1

CDFF apparatus, modified glass coverslip assembly, and imaging.

FIG. 2

Time-gated fluorescence lifetime imaging, in which the sample is excited with a brief excitation pulse, after which the fluorescence intensity is recorded in two (or more) time gates.

FIG. 3

Fluorescence penetration depth in xz sections of rhodamine B-stained biofilm by confocal (single-photon) imaging (a) and TPE (b). Depths are measured from the biofilm-coverslip interface.

FIG. 4

Axial (xz) intensity profiles derived from Fig. 3.

FIG. 5

Fluorescence xy images of 30 by 30 μm recorded at distances of 10, 40, and 60 μm from the biofilm-coverslip interface (see Fig. 1) by using CLSM (right) and TPE (left).

FIG. 6

Solid line, relation between biofilm pH and window ratio (W₁/W₄), with data points fitted to a Boltzmann sigmoidal curve: y = [(A₁ A₂)/(1 + e^{(x − x₅₀})/dx)] + A₂, where A₁ and A₂ are the asymptotes, x₅₀ is the center, and dx is the width (χ² = 3 × 10⁻⁵). The error bars reflect the variability (standard deviation) of three independent calibration experiments. Dashed line, relative fluorescence intensity of carboxyfluorescein as a function of pH.

FIG. 7

TPE xy images of biofilms at 5 μm (A) and 70 μm (C) from the biofilm-coverslip interface. (B) pH image of panel A. Bar, 5 μm.

FIG. 8

Window ratio (pH) image of an xz section through biofilm. A higher intensity (lighter image) in the window ratio images indicates a lower pH value. The top of the image is the biofilm-buffer interface; the bottom represents the biofilm-coverslip interface.

FIG. 9

Intensity (A and B) and window ratio (pH) (C and D) images in biofilm, before (A and C) and 60 min after (B and D) addition of a 14 mM sucrose solution. Images were recorded at 65 μm from the surface. The average pH values are 6.2 for panel C and 5.5 for panel D. Bar, 10 μm.

FIG. 10

Decrease in mean biofilm pH in a 100-μm biofilm following a sucrose pulse. The average pH over 30-by 30-μm xy images (100 by 100 pixels) at 70, 35, and 5 μm from the biofilm-coverslip assembly is shown.