Manfred Auer explains TEM microscope to Purbasha Sarkar (left) and Elena Bosneaga

Manfred Auer:

He’s the Man of the Hour for Mapping the Cell Wall

Since all of the action of breaking down lignin and other plant constituents to fermentable sugars will take place in the cell walls of second-generation biofuel feedstocks, it is probably a good idea to know the exact make-up of a cell wall. That complex challenge is being accepted by electron microscopist Manfred Auer and his team of imaging specialists at Lawrence Berkeley National Laboratory and on the UC Berkeley campus.

“We need a realistic model of a cell wall, to understand the ‘building principles’ that Nature has come up with,” said Auer, a staff scientist at Berkeley Lab. But due to its dense, complex, fibrous structure of cellulose, hemicellulose, pectin and lignin, built by about 1,000 proteins, getting a real image of it is extremely difficult. Cellulose microfibers are embedded in an amorphous matrix, like steel girders stabilizing a skyscraper.

Auer and his team are applying a process called “correlative microscopy,” which involves looking at a specific sample in three different ways and combining the resulting data into an overlay of 3-D architecture and composition. There’s much more to this than taking a photograph. “A model of a cell wall is not the same as a picture,” Auer says. “It has to be both representational and compositional. Our efforts will result in the first cell wall model that is based on actual 3D ultrastructural data, allowing us to obtain precise geometrical 3D measurements.”

It may sound fairly straightforward, but it’s not. The effort involves the combining of fluorescent tagging, laser light scattering (Raman microscopy), and three-dimensional electron microscopic data collection and reconstruction (electron tomography). As Auer notes, scientists don’t just want to know what chemicals are inside a material, which the Raman method will indicate. They want to know the exact positioning of the macromolecules in the cell wall. Such precision demands a three-dimensional molecular-resolution view

“Images are only as good as the sample preparation,” says Auer, and that’s where the expertise of Auer and his team comes in. A staff scientist in the Life Sciences Division at Berkeley Lab, Auer is in demand these days; EBI is one of 30 collaborations in which his advanced imaging approaches play a major role. The use of cutting-edge ultra-rapid high-pressure cryopreservation, followed by either low-temperature freeze-substitution of solvents for water, or cryo-sectioning in a sample’s frozen-hydrated state, are invaluable in the preparation of a piece of tissue or slice of an organism that can be “read” by various microscopes. The work proceeds at the nanometer scale, and to get to a single “image” can take a week or longer to complete.

Members of the team each bring different specialties. Kent McDonald, who directs the UC Berkeley electron microscopy lab, is an expert in sophisticated sample preparation. Ken Downing is the expert in cryo-electron tomography. Bahram Parvin will develop the computationally intensive image analysis platform, and Jan Liphardt is a wizard on Raman spectroscopy and optical microscopy.

Their initial subjects are two plants with established genetic records – Arabidopsis, a genus of the mustard family, and Brachypodium, a genus of grasses. But Auer said it could be most any plant – his goal is to image an “average” cell wall with a structure common to all plants.

“This (cell wall model) is something that will be useful to the entire (EBI) research community and beyond,” says Auer. “It is time to create an atlas of the plant’s cell wall structure. Such information is key for a rational design of plants that can be more easily broken down into fermentable sugars.”