APPLICATIONS OF TECHNOLOGY:
- Biotechnology – development of antibiotics
- Development of antifouling agents for martime vessels
- Biofuel development
ADVANTAGES:
- Real-time imaging of biofilm formation
- Uses living microbial cultures
- Continuous immunostaining
- Rapid results — experiments conducted in ~ 6 hours
- Multiscale observations
- Validated using model organisms
ABSTRACT:
Researchers from Berkeley Lab, UC Berkeley, and UC Santa Cruz have developed a novel system for direct, real time observation of bacterial formation of biofilms, the sticky plaques that shield many pathogens from antibiotics and play a significant role in human disease. Specifically, the team, led by Veysel Berk and Jan Liphardt, and including Steven Chu, combined an innovative continuous immunostaining technique with super-resolution light microscopy to produce the first stop-action video imagery of biofilm formation.
Classical immunostaining requires time consuming and potentially disruptive steps to stain cellular components, wash away unbound dyes or antibodies, and then take a single image “snapshot” of the labeled cells. The Berkeley Lab system carefully parcels out a minimal number of fluorescent tags to eliminate the need for washing, which can cause a signal-confounding background glow. Each tag is designed to bind to the surface of specific target molecule, and the gene expression and concentration of those targets is revealed by presence and strength of the glowing tags. With no need to interrupt processes for a washout step, the Berkeley Lab immunostaining technique can monitor gene activity continuously, in solution, so the sequence of biofilm building can be plotted accurately over time.
Confocal Laser Scanning Microscopes (CLSM) penetrate layers of microbial architecture though the use of extremely fine focus adjustments, but are restricted to the diffraction limit of light. The Berkeley Lab system uses a custom-built super-resolution light microscope (20 nm, a tenfold improvement over the 200 nm resolution of conventional light microscopy) in combination with CLSM, for exploring biofilms. In one experiment, this combination of advanced optics and staining techniques spotlighted the activities of the four gene products in V. cholerae biofilm formation over a period of six hours, and converted it into a 3-D animated, stop-action video that clearly shows the sequence of biofilm development.
An estimated 80 percent of microbial infections are associated with biofilms. Like body armor, these polysaccharide and proteinaceous shields make pathogens 1,000 times more resistant to antibiotics, confound attacks by the body’s immune system, and even resist cleaning by detergents. Understanding precisely how these microbial cities are constructed can yield clues on how to breach those defenses and chart a path toward more effective antibiotics or treatments for chronic disease such as cystic fibrosis. By producing real time, high-resolution images of bacterial behaviors at molecular scales, researchers can dissect and map the sequence of events that take place as biofilms are formed.
In addition, biofilms in the guts of termites play a crucial role in cellulose degradation, and understanding that process could lead to development of better biofuels. Biofilms that foul the bottoms of ships can reduce their energy efficiency by 20 percent. Understanding how this bio-fouling develops could lead to better anti-fouling agents that would improve shipping efficiency and reduce fuel consumption.
DEVELOPMENT STAGE: Bench scale prototype.
STATUS: Patent pending. Available for licensing or collaborative research.
FOR MORE INFORMATION:
SEE THESE OTHER BERKELEY LAB TECHNOLOGIES IN THIS FIELD:
Cells-on-a-Chip: Apparatus for Real Time, Label-free Molecular Imaging of Live Cells and Bacteria, JIB-2201
Proteins for Developing New Classes of Antibiotics, IB-2346
REFERENCE NUMBER: IB-3282
