Several fluorescence techniques have emerged recently that meet the challenge of detecting single molecules. The emphasis now is on observing molecular reactions. Because many important biomolecular functions occur in an aqueous environment, researchers at the University of Florida have developed a method for real-time imaging of individual molecules in solution that may enable the observation of chemical reactions of fluorescent molecules. The technique, developed by Xiaohong Fang and Weihong Tan of the university's department of chemistry, excites fluorophores with an evanescent wave field, characteristic of optical fiber. The researchers said that their method of achieving evanescent wave excitation is simpler and more flexible than the conventional prism method and should lead to practical applications for single-molecule detection. Evanescent excitation In their work, the excitation beam from an argon-ion laser from Coherent Laser Group of Santa Clara, Calif., is propagated through an immersed optical fiber from Newport Corp. of Irvine, Calif., and an evanescent electromagnetic field exits at the fiber-solution interface. The researchers focus a microscope objective on the outside of the optical fiber, and the collected signals are sent to a Princeton Instruments intensified charge-coupled device. Because the evanescent field penetrates only about 250 nm, only those fluorophores near the surface of the fiber are excited, and their fluorescent emissions are detected as single-pixel signals. Individual molecules appear as spots on the display. Researchers at the University of Florida have developed a method of imaging single molecules using evanescent wave excitation on the surface of an immersed optical fiber. "The thickness of the field is on the order of half the wavelength of the propagated light," said Fang. "In this way, we have a very small detection volume -- an important condition for single-molecule detection." She explained that the small detection volume reduces the background signal, which increases the signal- to-noise ratio. The researchers tested the system with fluorescein, rhodamine 6G and dye-labeled biomolecules. They achieved single-molecule detection by considering the volume, the dye concentration and the number of spots displayed in the images. In addition, an excellent linear relationship existed between fluorophore concentration and the number of pixel images. Fang said that the team is developing techniques to make the optical fibers more functional. "We think we can immobilize the molecules onto the surface of the optical fiber with such a low density that there is only one molecule in one detection unit. This may allow the optical fiber to act as a sensor or probe." The study is described in the Aug. 1, 1999, issue of Analytical Chemistry.