Visualizing Biofilms in Three Dimensions
Dr. Veysel Berk, University of California-Berkeley, Dr. Jiunn Fong, University of California-Santa Cruz, and colleagues
Many bacterial species can form biofilms, and these aggregates of cells are challenging to eliminate in medical, industrial, and natural settings. Researchers led by Dr. Veysel Berk and Dr. Jiunn Fong, of the University of California in Berkeley and Santa Cruz, respectively, have now visualized the molecular makeup and assembly of a biofilm using super-resolution and conventional light microscopy.
The researchers used the human pathogen Vibrio cholerae as a model organism to study the molecular mechanisms involved in biofilm development. The main biofilm constituents are matrix proteins, which are thought to maintain the biofilm mechanical stability, and Vibrio polysaccharide (VPS), which makes up the building blocks of V. cholerae biofilm. To investigate how these constituents interact during biofilm formation, the investigators generated red or green fluorescent V. cholerae strains that were unable to produce either matrix proteins or VPS. They then grew biofilms of these strains in flow cells.
This experiment revealed that V. cholerae strains lacking either biofilm matrix proteins or VPS cannot form mature biofilms. “We showed that when a strain of V. cholerae unable to produce biofilm matrix proteins is co-cultured with a V. cholerae strain unable to produce VPS, extracellular interaction of matrix components leads to mature biofilm formation,” Dr. Fong said.
Using a confocal laser-scanning microscope, they imaged the biofilms at 1-micron z intervals and then created 3D structures from the images using the Easy 3D function of Imaris. Because the two strains formed structured biofilms only in co-culture, it was important to capture the localization of the strains within the mixed biofilm (see figure). By using different color channels, they easily visualized the three-dimensional structures of each strain. “The user-friendly interface of the Imaris software allowed us to easily process and re-construct the 3D images,” Dr. Fong said.
The researchers also developed an in vivo labeling strategy that they used with super-resolution microscopy to visualize the extracellular matrix of developing biofilms and to identify the roles of the four essential matrix constituents. Their approach for deciphering the molecular architecture of biofilms in high-resolution and for imaging the assembly of biofilm components can be adapted to other bacterial species and help aid in development of anti-biofilm drugs. The researchers are now working to understand how the components of the biofilm matrix are generated and how they interact with one another.
Research Paper: Molecular architecture and assembly principles of Vibrio cholerae biofilms. Berk V, Fong JC, Dempsey GT, Develioglu ON, Zhuang X, Liphardt J, Yildiz FH, Chu S. Science. 2012 Jul 13;337(6091):236-9.