At the University of Pennsylvania in Philadelphia, scientists have shown that nanostructures can be observed with a resolution of 30 to 50 nm by combining many small pictures of meandering fluorescent single molecules. From these separate yet overlapping images of local properties and structures, the researchers have constructed a picture of the nanostructure as a whole.The technique combines sequential single-molecule images (a and b) into a composite image (c), formed using 500 frames. An atomic force microscopy image (d) also indicates the presence of a long, striplike defect, verifying the accuracy of the technique. Robin M. Hochstrasser, a chemistry professor at the university and a member of the research team, noted that the technique requires variations in the concentration of molecular probes. "The single-molecule images would be featureless if the molecules had a uniform spatial distribution," he said. Those variations should not be too large, however. Either too many or too few molecules would obliterate the image of the nanostructures.For the demonstration, the scientists created a nanostructure template, a polymer sitting on a glass slide. The template had features ranging from 300 to 1000 nm in height, as verified by atomic force microscopy. They capped this structure with another glass slide, creating a space through which a solution could percolate. They then employed total internal reflection microscopy using an Olympus microscope and appropriate filters to track the motion of TMR, a fluorescent probe from Molecular Probes Inc. of Eugene, Ore.For a light source, they used a 514.5-nm argon-ion laser from National Laser Co. of Salt Lake City, focusing it to a 30- to 85-µm spot size. By keeping below the critical angle of about 60°, they created total internal reflection, which confined fluorescence to a region near the interface of the cover slip and solution. They captured the single-molecule images using a camera from Roper Scientific to collect 20- to 50-ms exposures and merged them into a composite image of the nanostructure using software algorithms.The findings largely agreed with the atomic force microscopy results, but the single-molecule imaging was affected by the properties of the template. For instance, the researchers could see the effect of molecule absorption at reactive edges of the template.Hochstrasser said that the group recently used the method to image membranes. The goal, he added, is to visualize nanostructures such as membranes in biological systems -- especially in single live cells.