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AFM maps the diatom exoskeleton

BioPhotonics
Jun 2007
Hank Hogan

Diatoms form a hard exoskeleton that is a multilayer membrane highly organized from the nano- to the macroscale. Mimicking such nanostructured materials could be useful in drug delivery, filtration and photonics, but people cannot pull off what diatoms naturally do.

“This level of organization cannot be achieved by even the most sophisticated nanofabrication techniques available today,” said Nicolas Voelcker, an associate professor in the School of Chemistry, Physics and Earth Sciences at Flinders University in Bedford Park, Australia.

Voelcker was part of a team that recently used atomic force microscopy-based nanoindentation to map the mechanical properties of diatom membranes. Others in the group were from the Australian Nuclear Science and Technology Organisation in Sydney and the University of Chicago in Illinois. The scientists found information that might someday assist in mimicking the structure and that might eventually help provide answers about why diatoms have biosilica at all.

Diatom exoskeletons, or frustules, are constructed from silica — the main ingredient of sand — and are not weak, even though they are riddled with pores of various sizes. It takes hundreds of tons of force per square meter to break them, which likely protects them from predators. The fine features of frustules, which vary from species to species of diatom, have been studied extensively, but investigators had not previously linked high-resolution structural information with mechanical properties.

Previous research used a nanoindenter, which provides very precise hardness measurements but does not do so with a great deal of spatial precision. Thus, the exact location of the indentations was not known, and so the relationship between the structure and its mechanical properties was unclear.

In atomic force microscopy (AFM), a sharp tip mounted on the end of a cantilever scans across a sample. When imaging, the cantilever is forced to vibrate at its resonant frequency, and interactions with the sample’s surface change the vibration. An optical readout of the vibration is accomplished via a laser reflected off the cantilever’s end.

Mechanical properties

The same technique can probe the sample’s mechanical properties by forcing the tip down to make a nanoindentation and then measuring the cantilever’s deflection angle. As compared with a typical nanoindenter, there is a loss of hardness precision, but the researchers believed that this tradeoff was worth it because they thought that the technique could satisfactorily correlate the structural and nanomechanical properties and provide useful information for advanced manufacture of structures from nano- to macroscale, said team member Dusan Losic, who at the time was a postdoctoral researcher at Flinders.

The researchers used a Veeco AFM system with standard silicon tips mounted on the end of a standard cantilever. They also performed scanning electron microscopy (SEM) of the diatom frustules, coating them with a thin platinum film. Both AFM and SEM were done before any mechanical testing.

TSAFM1.jpg
The diatom, shown here in a scanning electron microscope image, builds nanostructures out of sand. Using an atomic force microscope, researchers performed nanoindentations at the locations indicated by the white squares to determine the strength and mechanical properties of the layers of a diatom’s biosilica. The cribellum (top) and the girdle bands (bottom) were the weakest. Images reprinted with permission of Langmuir.

For nanoindentation, they drove a diamond tip that was mounted on the end of a stainless steel cantilever into the surface at the desired location in incremental force steps until a predetermined deflection was reached. From the known springiness of the cantilever, they calculated the force exerted on the surface and its hardness.

They forced the tip in single instances and in an automated fashion, with up to 10 indents being done on various frustule layers at different locations. They followed the surface testing with another image scan, this time using the diamond tip with low applied force.

The researchers mapped the structure and mechanical properties of the Coscinodiscus sp. diatom because, as one of the largest genera of marine planktonic diatoms, it has diameters up to a few hundred microns and could be grown easily in a lab. For a comparison, they also tested the mechanical properties of porous silicon films and free-standing membranes.

They found that the hardness and elastic modulus varied in different parts of frustules, with the weakest being the cribellum and girdle bands that join the two halves of a diatom — the top and bottom. These weakest areas had roughly a tenth the hardness and a fourth the elastic modulus of the strongest and innermost layer, the internal plate. The work was published in the April 24 issue of Langmuir.

TSAFM2.jpg
The AFM image (a) shows nanoindentations on the cribellum surface, with the inset showing a zoomed view of the dashed line. Below is a typical force penetration curve showing loading (dashed arrow going up) and unloading (dashed arrow going down) as the AFM is forced through the surface (b). The solid arrows show kinks in the loading where something changed.

Cribellum and girdle bands

The weakness of the cribellum was expected. SEM imaging had determined it to be the thinnest pore layer and to have the smallest pores. The second finding was a surprise. The researchers had thought that the girdle bands would have about the same mechanical strength as the top of the diatom. Also unexpected was the finding that the cribellum was similar in hardness to porous silicon.

Losic noted that improving the software to streamline data processing for calculation of hardness and elastic modulus and to allow spatial mapping of nanomechanical properties would be helpful for this type of study.

As for the future, the researchers are looking into a variety of areas. One involves improvements in the reliability of AFM nanoindentation, which could involve changes to the tip, the cantilever and the piezoelectric scanner, and to their calibration. They also are considering a comparative study of the micromechanical properties of porous silicon using both AFM and a commercial nanoindenter. And they are examining the photonic and optical properties of live diatoms and diatom frustules.

Voelcker reported having some interesting results and hopes to collaborate with research groups that have photonics expertise on the biosilica. “It is simply the most fascinating nanostructured biomineral.”

Contact: Nicolas Voelcker, Flinders University; e-mail: nico.voelcker@flinders.edu.au.


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