Super-resolution microscopy highlights bacterial biofilms

By MedWire Reporters

A multi-fluorescent labeling strategy combined with super-resolution light microscopy has allowed researchers to observe the development of a bacterial biofilm in realtime.

The team watched the development of the Vibrio cholerae biofilm with "single-protein and single-polymer precision, revealing assembly principles and intermediates."

"The cells organize into clusters within the biofilm and the mature biofilm is a composite of these clusters," report Veysel Berk (University of California, Berkeley, USA) and colleagues in Science.

The researchers explain that 99.9% of all bacteria attach to surfaces, often creating biofilms, and that 80% of all infections in humans are related to biofilms.

Biofilms, they add, can be 1000 times more resistant to antibiotics and are typically removed surgically.

Prior to this study, bacterial communities were studied in terms of their average composition, appearance, and bulk biochemistry. The mechanisms in which the proteins and polysaccharides combined to form a biofilm were largely unknown.

The introduction of the in vivo fluorescence tagging strategy allowed the researchers to visualize the molecular and architectural roles of matrix proteins and extracellular polysaccharides of a growing V. cholerae biofilm.

The group developed a technique known as "continuous immunostaining" that allowed tracking of four different molecules using fluorescent dyes.

Multiresolution imaging of the living V. cholerae biofilms showed a complementary role of four essential matrix constituents.

The matrix protein RbmA was responsible for cell-to-cell adhesion, while Bap1 allowed the developing biofilm to adhere to surfaces. A mixture of the Vibrio polysaccharide, the matrix protein RbmC, and Bap1 combined to form the "dynamic, flexible, and ordered envelopes that encased the cell clusters."

The cell clusters are separated by microchannels that allow nutrients to enter and waste products to exit.

The super-resolution light microscopy is capable of resolution 10 times higher than standard microscopy, highlighting only a part of the image at a time using photo-switchable probes and compiling thousands of images into a single snapshot.

"The classical approach is first staining, then destaining, then taking only a single snapshot," Berk said in a press release. "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."

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