Bacteria also form permanent, mostly lifelong, biofilms in the mucus-filled lungs of cystic fibrosis patients and are responsible for the chronic lung infections that lead to early death. Although long-lasting antibiotic treatment helps, it cannot eradicate the infection completely.
To study a biofilm formed by cholera bacteria (Vibrio cholerae), Berk built his own super-resolution microscope in the basement of UC Berkeley’s Stanley Hall based on a 2007 design by coauthor Xiaowei Zhuang, Chu’s former post-doctoral student who is now a professor at Harvard University. To actually see these cells as they divided to form “castles,” Berk devised a new technique called continuous immunostaining that allowed him to track four separate target molecules by means of four separate fluorescent dyes.
He discovered that, over a period of about six hours, a single bacterium laid down a glue to attach itself to a surface, then divided into daughter cells, making certain to cement each daughter to itself before splitting in two. The daughters continued to divide until they formed a cluster – like a brick and mortar building – at which point the bacteria secreted a protein that encased the cluster like the shell of a building.
The clusters are separated by microchannels that may allow nutrients in and waste out, Berk said.
“If we can find a drug to get rid of the glue protein, we can move the building as a whole. Or if we can get rid of the cement protein, we can dissolve everything and collapse the building, providing antibiotic access,” Berk said. “These can be targets for site-specific, antibiotic medicines in the future.”
Super-resolution microscopy: painting with light
Berk is a biologist trained in physics and optics with expertise in imaging the structures of proteins: He was part of a team that a few years ago determined the atomic-scale structures of the ribosome, the cellular machine that translates genetic message into a finished protein.
He suspected that powerful new super-resolution light microscopy could reveal the unknown structure of biofilms. Super-resolution microscopy obtains 10 times better resolution than standard light microscopy – 20 instead of 200 nanometers – by highlighting only part of the image at a time using photo-switchable probes and compiling thousands of images into a single snapshot. The process is much like painting with light – shining a flashlight beam on a dark scene while leaving the camera shutter open. Each snapshot may take a few minutes to compile, but for slow cellular growth, that’s quick enough to obtain a stop-action movie.
The problem was how to label the cells with fluorescent dyes to continuously monitor their growth and division. Normally, biologists attach primary antibodies to cells, then flood the cells with fluorescent dye attached to a secondary antibody that latches onto the primary. They then flush away the excess dye, shine light on the dyed cells and photograph the fluorescence.
Berk suspected that a critically balanced concentration of fluorescent stain – low enough to prevent background, but high enough to have efficient staining – would work just as well and eliminate the need to flush out excess dye for fear it would create a background glow.
“The classical approach is first staining, then destaining, then taking only a single snapshot,” Berk said. “We found a way to do staining and keep all the fluorescent probes inside the solution while we do the imaging, so we can continuously monitor everything, starting from a single cell all the way to a mature biofilm. Instead of one snapshot, we are recording a whole movie.”
“It was a very simple, cool idea, but everyone thought it was crazy,” he said. “Yes, it was crazy, but it worked.”
Berk’s coauthors are Steven Chu, now with the U.S. Department of Energy; Jan Liphardt, UC Berkeley professor of physics and of molecular and cell biology; Xiaowei Zhuang and Graham T. Dempsey of Harvard; Jiunn C. N. Fong and Fitnat H. Yildiz of UC Santa Cruz; and Omer N. Develioglu of Taksim Research Hospital in Istanbul, Turkey.