LEIDEN, THE NETHERLANDS--Researchers can map single atoms or molecules on surfaces almost as routinely as cartographers map hills and lakes, thanks to instruments like the scanning tunneling microscope. But below the surface, they start to lose their bearings. In the 31 July issue of Chemical Physics Letters, however, physicists from the universities of Leiden and Amsterdam in the Netherlands bring new accuracy to subsurface molecular imaging--work that could open the door to three-dimensional mapping of the cell.
Molecular imaging has been hampered by the diffraction limit, an intrinsic blurring of light that prevents two sources from being resolved when they are close together. In 1995, Eric Betzig, of NSOM Enterprises, proposed a way to get around the diffraction limit. Each molecule in a solid matrix finds itself in a slightly different structural environment because of random strains and imperfections. As a result, each one has an absorption line at a slightly different frequency. This shift is generally very small, but at low temperatures it can easily be resolved with a tunable laser that generates a precise frequency of light. "Molecules which can normally not be spatially separated are clearly distinguished when spectral selection is applied," explains Jürgen Köhler of Leiden University.
Köhler and his colleagues illustrated the method on a sample of pentacene, an aromatic hydrocarbon, in a host crystal of p-terphenyl. Pentacene fluoresces strongly when excited by laser light. By moving the focus of the laser through the sample in three dimensions and determining the position of the fluorescence maximum for each molecule, the group could pinpoint its location with an accuracy well below the diffraction limit.
Köhler and his colleagues, says Niek van Hulst of the University of Twente in the Netherlands, "are pushing optical microscopy to its limits." The technique might uncover new details about the inner workings of cells. Researchers might, for example, label genes with different fluorescent molecules, then determine the precise positions of these marker molecules to learn, say, how the DNA in chromosomes twists and coils.