A new technology could provide nanometer-resolution, 3-D images of small semiconducting structures or proteins inside cells, according to a proposal in the 20 November Physical Review Letters. The system would construct images by examining how light scatters off of the sample and a nearby metal nanoparticle positioned with an atomic force microscope. The nanoparticle solves a crucial problem faced by an earlier design and may allow the technique to become a practical way of imaging small objects without cutting into them.
Researchers would like to see inside small structures, such as a bundle of proteins inside a cell membrane, without disturbing their sample. Several techniques for imaging cells involve fluorescence and give mainly a 2-D picture. Other techniques use visible light to see details as small as tens of nanometers but are currently limited to surface imaging. For example, in one version of a near-field scanning optical microscope (NSOM), a hair-thin optical fiber is scanned very close to an illuminated sample to detect scattered light. This “near field” light contains information about the surface structure at scales smaller than a wavelength of light.
To see beneath the surface, John Schotland of the University of Pennsylvania in Philadelphia and his colleagues previously suggested a method that also uses the near-field. In their set-up, a sample, thinner than a wavelength of visible light, is placed on top of a glass prism. A beam of laser light shines on the sample from below, through the prism. The light bounces off the boundary between prism and sample, where some of it scatters upward, illuminating the sample, and the rest reflects back down onto a detector. But in order to extract the structural details from the reflected light, the experimenters need to either control where the peaks and troughs occur in the incident beam, or detect where they occur in the reflected beam–that is, they need to control or detect the light’s phase. In experiments, this proved too difficult.
Now Schotland and his colleagues have proposed a new method, using a similar setup, that eliminates the need for phase information and instead uses an atomic force microscope (AFM), a device famous for its precise maneuverability. First, a tiny object that scatters light strongly, such as a gold nanoparticle, is attached to the AFM’s slender probe, which then hovers less than a wavelength above the sample’s surface. Next, aiming the laser at the prism-sample interface from below, a researcher would move the AFM’s probe delicately over the sample’s surface while a detector measured the power of the reflected beam. “The atomic force microscope has the capability of changing the position of the tip [at] nanometer scales with extraordinary precision,” Schotland says.
The location of the nanoparticle and the structure of the sample determine the amount of light absorbed at the prism-sample interface, and therefore the power reflected back to the detector. Based on the power detected for every position of the gold nanoparticle, the technique can extract the structure of the sample at different depths, creating a three-dimensional picture.
Schotland says the method could see details as small as ten nanometers, such as a large protein on the surface of a cell. Deep inside the sample, the method could pick out objects as small as tens of nanometers, such as a ribosome, an organelle found inside cells. Unlike other methods that literally slice a sample to get at inner details, the new method is non-invasive, says Schotland, and it uses readily-available tools.
Lukas Novotny of the University of Rochester calls the proposed method “a truly novel approach to near-field microscopy” with a potentially “big impact” on researchers’ ability to characterize tiny structures. On the other hand, he imagines several experimental challenges, such as calibrating the system with known samples. “Ultimately the proposed scheme has to be validated by experiments,” he says.