Super-resolution microscopy shows details of biofilm structure
A new imaging technique discovered at the University of California, Berkeley, reveals a possible plan of attack for many bacterial diseases.
By devising a new fluorescent labeling strategy and employing super-resolution light microscopy, researchers were able to examine the structure of bacterial biofilms.
They also identified genetic targets for potential drugs that could break up the bacterial community and expose the bugs to the killing power of antibiotics.
“Eventually, we want to make these bugs homeless,” said lead researcher Veysel Berk, a postdoctoral fellow in the Department of Physics and the California Institute for Quantitative Biosciences (QB3) at UC Berkeley
Berk 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.”