AFM: Not Just for Materials Science Anymore

CANTILEVERS AND TIPS

Paul Hansma of UC-Santa Barbara is widely acknowledged as the dean of AFM development. "The family of cantilever-based, scanning-probe microscopes is generically called atomic-force microscopes, in honor of the name given them by the inventors of the first such instrument," says Hansma.¹ "In order to help people with literature searches, [referees try] to keep the name AFM ... despite billions of variations you can do with cantilever-based instruments."

A cantilever is like a tiny diving board, made of silicon or silicon nitride, with an upside-down pyramid-shaped tip at the end. The cantilever's dimensions set the force resolution, while the tip's radius governs the imaging resolution. The system noise floor (inherent noise) is also a limiting factor.

"Any noise that contributes to cantilever deflection is a fundamental limit on the force that you can resolve or on the scale of the image that you can resolve," says Matthew Thompson, life sciences development manager in the Nanobio Instruments Group at Veeco Instruments of Woodbury, NY. Whether the noise source is acoustic or mechanical, anything that causes additional change between the head (which holds the tip) and the sample is going to translate into changes in the cantilever deflection.

The microscope's probe (cantilever and stylus tip) scans the sample in raster fashion: across, down, and across again, like an old dot-matrix printer. Raster comes from the Latin for rake, which is a fairly accurate approximation of what actually happens during a scan: in certain modalities the tip makes continuous contact with the sample, in others (tapping mode) it touches down intermittently. "One of the advantages of the AFM is it's a kind of 3-D microscopy – you measure X, Y, and Z, so you can measure real heights of features," says Hansma.

The force applied on the surface by the cantilever as it flexes over the surface is given by Hooke's Law, F = ks, where F is applied force, s is the deformation of the body caused by that force, and k is a spring constant. The cantilever's deflection (e.g., s) is most commonly detected through the beam-bounce method, in which a laser beam, bounced off the tip of the cantilever, hits a detector. When the cantilever deflects, the beam's movement is detected with a split photodiode detector, which emits an electrical signal that is proportional to the extent of the deflection.

Though most biologists use AFMs for imaging, the biophysical community has adapted them for use in force-spectroscopy experiments. "It's essentially the same instrument," says Calvin Quate of Stanford University, who, with Gerd Bennig and Christoph Gerber, invented the first AFM. "It's modified in the sense there's no scanning required, the tip just dips down and picks up a protein molecule, and lifts back and stretches it and it unfolds, and they can monitor the unfolding. Then they stretch it until it breaks, and they get some idea of the [molecule's] elastic properties." (Veeco Instruments' 30-page A Practical Guide to SPM,² lists four primary imaging modes, 11 secondary imaging modes, and nine nonimaging modes.)

The tip can also be biofunctionalized for various kinds of experiments, says Patrick Collier of the California Institute of Technology. For example, Collier cites dippen nanolithogra-phy, Chicago-based NanoInk's 21st-century advance on, well, inkwells. "You coat the AFM tip with molecules (your 'ink') and dip the tip into the substrate (your 'paper')," he explains.

Or, suggests Fred Sachs of the University at Buffalo, NY, "You could put a specific ligand on the tip, if you're looking for a neuro-transmitter receptor .... You go poking around in this raster, wherever it sticks, you find a high force, and wherever it doesn't stick, you find a low force, and you could make a map of where the physical location of receptors are."

ONE DRAWBACK, SEVERAL SOLUTIONS

AFMs have one well-known weakness, says Michael Roukes of Caltech: "The time it takes to acquire an image." They typically scan almost 30 times slower than an electron microscope. As a result, certain biological events, such as nuclear pore-complex activity or lipid thermodynamics and other high-speed dynamic processes, cannot be captured using current AFMs.

That wouldn't be the case, however, if the cantilevers were made smaller than the current 100 microns (see sidebar). "The resolution limit that we have is fluctuations in the cantilevers," says Hermann Gaub of Ludwig-Maximilian University of Munich. "The smaller a cantilever is, the less it dissipates when it moves through liquid, and if it has less dissipation, it will also have less fluctuation."

"There's a lot more to the instrument than the cantilevers," says Thompson. For optimal performance, the feedback loop and actuator should also be adjusted to follow the much higher changes on the sample surface detected by smaller cantilevers.

But, when small cantilevers are fully developed, there may still be a Catch-22 in terms of costs. Typically you clip a cantilever into the microscope. But current AFMs are tailored to standard-size cantilevers, and after a certain shrinkage, it's no longer possible to simply swap-in new cantilevers. Both commercial and academic sources say next-generation commercial AFMs will likely address this problem.