Combined TIRF data overlaid on AFM topography.Localization of actin filaments relative to the surface topography of singlecells (HeLa). Imaged with a custom-built combination of an Asylum Research MFP-3DAFM and a TIRF microscope constructed on an Olympus IX81. Topographic and fluorescencedata were collected simultaneously, 40µm scan. Image courtesy of MiklósS.Z. Kellermayer, Nanobiotechnology and Single-molecule Biophysics Group, Univ.of Pécs, Hungary.
Invented in 1986, the atomic force microscope (AFM) is finally coming into its own, taking on an active role in electronics, materials and life science research. While it remains slower than optical microscopy, the AFM isn’t affected by the inherent resolution limits of light optics, and so can givetrue atomic-level information, yielding a true three dimensional surface profile.
When compared to other methodologies, the benefits are clear. In contrast to scanning tunneling microscopes (STMs), which send current to the surface being observed, AFMs can be used to image non-conductive materials. In comparison to electron microscopes, AFMs typically can image samples in their native environments, require much less demanding sample preparation, and allow direct probing ofsuch nanoscale surface properties as elasticity and conductivity.
But until recently, the AFM seemed more of a novelty than a laboratory workhorse. “It’s almost like we are where transmission electron microscopy (TEM) was 40 years ago,” says Vance Nau, President and CEO of Molecular Imaging Corp., Tempe, Ariz. “Until a few years ago, we were mainly exploring the methodology itself, almost more to discover what it wascapable of than to solve real problems.”
Craig Prater, Director of Technology Development for Veeco Instruments Inc., Woodbury, NY agrees. “Over the last several years, the AFM has moved from a laboratory curiosity to one of the fundamental tools for studying life science specimens, semiconductors, polymers, biomaterials, and much more.” Although hard numbers are not available, most studies indicate that the number of AFM systems in the field is now close to 10,000--more than double the number justa few years ago.
Faster than a speeding…
In the past year alone, vast leaps have been made in the capabilities and range of AFMs. The first of these advances is in the area of speed. AFMs have a cantilever with a very narrow tip at one end that is brought close to a sample surface, and is deflected according to Hooke’s law. Typically, the deflection is measured using a laser spot that is reflected from the cantilever to a detector. The pattern of vertical displacements results in a map representing the topography of the sample. The AFM can record images with extraordinary spatial resolution. However, data must be collected sequentially as the tip moves, sometimes requiring many seconds to collect sufficientinformation for meaningful analysis.
“Sequential imaging can be very powerful,” said Molecular Imaging’s Nau. “Think of all the information that can be communicated through touch--heat, electrical information, data on surface rigidity or other properties, and more. But this kind of data can take time to acquire. This has been one of the greatchallenges to manufacturers.”
But that may be changing. All of the key AFM companies say that their instruments are getting faster. And recently, scientists from the Massachusetts Institute of Technology, Cambridge, the Univ. of Calif., San Francisco, and Israel’s Weizmann Institute of Science, Rehovot, demonstrated an AFM that can capture images of dynamic processes with a time resolution of microseconds. Moshiur Anwar and Itay Rousso have developed a technique that involves breaking down a sample into individual pixels and measuring the dynamics of each pixel separately with the AFM. According to the researchers, this “step scan” technique can resolve features 10 nm wide with a time resolution of 5 msec--an order of magnitude faster than is possible with the usual “rapid-scan” method.This technique only works for periodic processes so far.
Cat Tail Collagen Fiber. Images captured using Molecular Imaging's PicoPlus AFM. 2a)
Top - AAC (Acoustic Alternating Contact) Mode Image was obtained in air. 3.3 µm × 3.3 µm. 2b)
Bottom - MAC (Magnetic Alternating Contact) Mode image was obtained in water after re-hydration of the collagen sample. 1.4 µm × 1.4 µm. (Courtesy of Molecular Imaging).
Wet and wild
A second limitation addressed in recent science is the difficulty in using AFMs for viewing cells, tissues or materials that must be kept alive and immersed in liquid during observation. But life science observations in waterhave now become a powerful area of AFM research.
“The first AFM that involved imaging under liquid goes back to the 1990s,” says Veeco’s Prater. “Researchers are now using AFM to obtain high-resolution images of human viruses, for instance, or to image the opening and closing of pores in a cell’s surface as it takes in materialfrom its environment, for example.”
Scientist Jim Gimzewski of the Univ. of Calif., Los Angeles, whose prior work included a molecular “propeller” and the world’s smallest abacus fashioned out of buckyballs (a type of carbon molecule), last year used a Veeco BioScope AFM to study the way yeast cell walls vibrate with a “pulse” that has a detectable hum. His conclusion--that yeast cells generate a type of sound through these vibrations--have led to a new area of AFM research, into sono-cytology,or the audio characteristics of cells.
Two methodologies in one
One of the most exciting areas of development in the AFM arena is in combining it with other research techniques for investigating a single phenomenon, allowing researchers to benefit from the advantages of using different methodologiessimultaneously.
“The importance of combined functionality is enormous,” says Roger Proksch, president of Asylum Research, Santa Barbara, Calif., which has brought systems that combine confocal optical imaging and AFM to market. “Software is one of the keys to making such a combined system work, and it presents some inherent challenges. But the fact that you can collect and assemble complementary data to observe and learn new things that neither technique alone could give you is vital.” Hungarian scientist Miklós Kellermayer, for instance, uses one of the company’s AFM systems together with confocal and total internal reflection fluorescence (TIRF) optical microscopy systems to stimulateand then map calcium ion release in living cells in real time (Fig. 3).
Molecular Imaging also recently introduced its dual-function PicoTREC system, which adds real-time, simultaneous chemical imaging capability to an AFM. By providing simultaneous topography and recognition information with single molecule sensitivity and without radioactive, fluorescent or other markers, it can be used to map molecular binding and adhesion events on surfaces. With applications in semiconductor mask-making and wafer fabrication, data storage, drug discovery and polymer research, it has the potential to help researchers better understandthe structures and interactions that influence molecular-level events.
Digital Instruments EnviroScope AFM piezoresponse image of BiFeO3 spheroidal structures on SrTiO3 substrate. Light and dark shades correspond to ferroelectric polarization oriented up/down. 5 micron scan courtesy of C. Harnagea, C. Cojocaru and A. Pignolet, INRS-EMT/Univ. of Quebec, Canada.
Another two-in-one approach was recently described by Qiling Tang and his colleagues from the Illinois Institute of Technology and Argonne National Laboratory. They have combined AFM, bioconjugation and fluorescence microscopy to open up the possibility of monitoring protein functions in cells in real time. Using their system, they delivered proteins to a surface with nano-scale precision, examined the region immediately, and confirmed the reproducibility of their findings with fluorescence imaging. Importantly, protein delivery and subsequent imaging were performed in the same local area with the same AFM tip, so it appears possible that protein functions in living cells could one day be monitoredin real time using a similar system.
“There is so much information that couldn’t possibly be acquired without the additional channels,” says Asylum Research’s Proksch. “When you add that to the important potential that the AFM has for such fields as nanolithography, polymer science, data storage and biological applications, it is no wonder the field is growing so rapidly. We still have so many fundamental scientific questionsto answer.”
— Ilene K. Semiatin
Ilene Semiatin is a freelance writer
based in White Plains, N.Y.