Although scientists commonly use far-field polarization microscopy to measure the orientation of single molecules, it was thought that the technology could obtain only two-dimensional information. Now researchers at the Massachusetts Institute of Technology have demonstrated the technology's ability to measure the 3-D orientation of highly symmetric single chromophores. "With this technique, relatively low-resolution optical microscopy can be used to get this very detailed information about a structure that is many orders of magnitude smaller than the diffraction limit of light," said Stephen A. Empedocles, a researcher on the project who is now a scientist with biotech start-up Quantum Dot Corp. in Palo Alto, Calif. "In our field, that's opened up a lot of new areas of research." Described in the May 13 issue of Nature, the research was done with CdSe nanocrystal quantum dots, which act like large single atoms. Speed and simplicity Although other techniques -- such as near-field scanning optical microscopy -- can measure the 3-D orientation of chromophores, the method that the MIT team describes measures as many as 500 single nanocrystals in 60 seconds, a time that could be reduced to less than 0.1 s with some minor modifications, according to Empedocles. The microscope was designed for high light-detection efficiency. It included a Coherent argon-ion laser; a Nikon objective lens with a high numerical aperture; and a liquid-nitrogen-cooled, back-thinned charge-coupled device camera from Princeton Instruments. "The entire experiment consists of inserting a linear polarizer in either the excitation or emission pathway and rotating it," Empedocles said. "If a person has the ability to detect single molecules, the amount of effort to do this procedure is almost zero." The speed of gathering data is limited only by the number of photons emitted by each chromophore, he added. There is another limit: To measure the full 3-D orientation, the molecule must emit light that is polarized isotropically in a plane, which generally requires a threefold or higher rotational symmetry. "We did our study on nanocrystallites, but -- in theory -- this could be done for any chromophore with the appropriate symmetry," Empedocles said.