Optically transparent porous medium for nondestructive studies of microbial biofilm architecture and transport dynamics

Abstract

We describe a novel and noninvasive, microscopy-based method for visualizing the structure and dynamics of microbial biofilms, individual fluorescent microbial cells, and inorganic colloids within a model porous medium. Biofilms growing in flow cells packed with granules of an amorphous fluoropolymer could be visualized as a consequence of refractive index matching between the solid fluoropolymer grains and the aqueous immersion medium. In conjunction with the capabilities of confocal microscopy for nondestructive optical sectioning, the use of amorphous fluoropolymers as a solid matrix permits observation of organisms and dynamic processes to a depth of 2 to 3 mm, whereas sediment biofilms growing in sand-filled flow cells can only be visualized in the region adjacent to the flow cell wall. This method differs fundamentally from other refractive index-matching applications in that optical transparency was achieved by matching a solid phase to water (and not vice versa), thereby permitting real-time microscopic studies of particulate-containing, low-refractive-index media such as biological and chromatographic systems.

FIG. 1.

Refractive index matching as demonstrated with a constant solid phase (glass bead) and various common immersion media: water (A), 80% glycerol (B), and microscopy immersion oil (C), with refractive indices (RI) as indicated. The bead has a refractive index of approximately 1.55 and therefore is almost completely transparent in immersion oil (as shown by color similarity); note, however, that light transmission is attenuated significantly by the immersion medium (implying higher absorption), as indicated by the lighter background color compared to that in panel A (for example). Scale bar, 200 μm.

FIG. 2.

Optical properties of commonly used porous medium materials and Nafion. (A) Photographic target measuring 2 by 2 cm; (B) dry porous media in rectangular glass capillaries; (C) porous media immersed in distilled water. Note that the optical target behind the Nafion-filled capillary becomes visible after filling with water but remains essentially free from distortion. (D) Preservation of signal intensity observed for multiple layers of Nafion grains (generally limited to three layers by the geometry of the rectangular capillary used for microscopy). The color profile refers to an arbitrary intensity scale, whereby similar colors represent similar transmission properties. (E) Optical image and transmission profile corresponding to data shown in panel D. Gain settings were increased to make the Nafion visible. Scale bars (D and E), 200 μm.

FIG. 3.

Comparison of refractive index matching and intensity profiles for water-immersed conventional porous media and Nafion by confocal laser scanning microscopy. (A and B) Individual sand grains and glass beads, respectively, demonstrating considerable refractive index mismatch with the surrounding medium; (C) multiple layers of Nafion grains. Data shown in panels A to C were recorded at a lower detector gain setting. (D to F) Multiple layers of sand, glass beads, and Nafion, respectively, visualized at typical gain settings; (G to I) optical images corresponding to data shown in panels D to F and their intensity profiles for the regions indicated by red lines; the intensity for panel I is at the arbitrary maximum over the entire line profile, as indicated by the arrow in the graph at the bottom right of the panel. Note also the fine material present in the backgrounds of panels A and B. All scale bars, 200 μm.

FIG. 4.

Refractive index values obtained for dry and hydrated polymer films.

FIG. 5.

Three-dimensional reconstruction of a P. aeruginosa SG81 biofilm growing on a Nafion grain in a flow cell. Unlike the case for conventional sand columns, the biofilm is visible in its entirety on individual grains, as shown within the depths of the flow cell. The surrounding grains were removed electronically for clarity.

FIG. 6.

Visualization of P. aeruginosa SG81 biofilm formed on Nafion grains after being stained with the Live/Dead cell membrane permeability kit (see Materials and Methods for details). Red cells are stained with the cell-impermeant dye propidium iodide and therefore have compromised membranes, implying lack of viability.