Three-Dimensional Structure of Biofilm Formed on Glass Surfaces Revealed Using Scanning Ion Conductance Microscopy Combined with Confocal Laser Scanning Microscopy

Abstract

Biofilms cause a variety of problems, such as food spoilage, food poisoning, infection, tooth decay, periodontal disease, and metal corrosion, so knowledge on biofilm prevention and removal is important. A detailed observation of the three-dimensional structure of biofilms on the nanoscale is expected to provide insight into this. In this study, we report on the successful in situ nanoscale observations of a marine bacterial biofilm on glass in phosphate buffer solution (PBS) using both scanning ion conductance microscopy (SICM) and confocal laser scanning microscopy (CLSM) over the same area. By observing the same area by SICM and CLSM, we were able to clarify the three-dimensional morphology of the biofilm, the arrangement of bacteria within the biofilm, and the difference in local ion conductivity within the biofilm simultaneously, which could not be achieved by observation using a microscope alone.

Keywords: Aliivibrio fischeri; biofilm; confocal laser scanning microscope; in situ observation; scanning ion conductive microscope.

Figure 1

Schematic diagram of biofilm formation.

Figure 2

Outline of the SICM.

Figure 3

Three locations were observed using SICM: one where the biofilm was barely visible in the optical microscope image (b), one where a thin biofilm was visible (c), and one where a thick biofilm was visible (d). The optical microscope image is shown in (a). (e) shows the SICM image obtained at the same location for (d) after a rough scan (32 × 32 pixel) applied at negative bias. The images in (b–e) are obtained at 128 × 128 pixels. Comparing (d) and (e), the approximate shapes in the XY directions are the same, but parts that could not be detected in (d) are evident in (e), especially in the area surrounded by the white dashed line.

Figure 3

Three locations were observed using SICM: one where the biofilm was barely visible in the optical microscope image (b), one where a thin biofilm was visible (c), and one where a thick biofilm was visible (d). The optical microscope image is shown in (a). (e) shows the SICM image obtained at the same location for (d) after a rough scan (32 × 32 pixel) applied at negative bias. The images in (b–e) are obtained at 128 × 128 pixels. Comparing (d) and (e), the approximate shapes in the XY directions are the same, but parts that could not be detected in (d) are evident in (e), especially in the area surrounded by the white dashed line.

Figure 4

Biofilms observed using (a) CLSM and (b) SICM. The ion current threshold value for SICM observation was set to 1.0%.

Figure 5

Biofilms observed using CLSM at (a1) 0–5 µm, (a2) 5–8 µm, and (a3) 8–11 µm from the substrate and those observed using SICM at (b1) 0–5 µm, (b2) 5–8 µm, and (b3) 8–11 µm from the substrate. Comparing (a1) and (b1), the shape of the biofilm and the arrangement of the bacteria are consistent. Biofilm in the part surrounded by a dashed yellow circle on the upper right is not detected in either (a2) or (b2). The areas within the dashed yellow circles on the left in (a2) and (b2) are fluorescent in CLSM but cannot be detected using SICM. Comparing (a3) and (b3), the part surrounded by a dashed yellow circle can be detected using SICM but does not fluoresce with CLSM.

Figure 5

Biofilms observed using CLSM at (a1) 0–5 µm, (a2) 5–8 µm, and (a3) 8–11 µm from the substrate and those observed using SICM at (b1) 0–5 µm, (b2) 5–8 µm, and (b3) 8–11 µm from the substrate. Comparing (a1) and (b1), the shape of the biofilm and the arrangement of the bacteria are consistent. Biofilm in the part surrounded by a dashed yellow circle on the upper right is not detected in either (a2) or (b2). The areas within the dashed yellow circles on the left in (a2) and (b2) are fluorescent in CLSM but cannot be detected using SICM. Comparing (a3) and (b3), the part surrounded by a dashed yellow circle can be detected using SICM but does not fluoresce with CLSM.

Figure 6

Biofilms observed two-dimensionally using (a) CLSM and (b) SICM. The ion current threshold value for SICM observation was set to 2.0%. Red line corresponds to ZX plane in Figure 7. Dashed white square (i–iv) corresponds to the location where the XY plane was observed in Figure 7(ai,aii,biii,biv).

Figure 7

ZX plane images of biofilms observed using (a) CLSM and (b) SICM. The threshold value for the ion current for SICM observation was set to 2.0%. When the images in (i) and (iii) are considered together, a location where a large amount of ionic current can flow between the bacteria and the substrate can be detected. When the images in (ii) and (iv) are interpreted together, a place where a smaller amount of ionic current can flow between the bacteria and the substrate can be detected.