9604382 So The development of single molecule imaging and spectroscopy technology has the potential to revolutionize the study of proteins. One of the most promising approaches utilizes two-photon excitation. By focusing a high peak power laser to a diffraction limited spot, chromophores can be effectively excited by the simultaneous absorption of two photons each having half the energy needed for the excitation transition. Because of the high power density requirement, the two-photon effect is confined to a sub-femtoliter volume at the focal point. For single molecule study, this localization ensures that the ubiquitous background fluorescence does not overwhelm the fluorescence from a single protein molecule. Compared with other approaches, two-photon excitation has the added advantages that Raleigh and Raman scattering can be easily eliminated, and proteins outside the excitation volume will not be photobleached. Further, the 3-D confinement of the two-photon excitation volume offers the opportunity to study these protein molecules in their natural aqueous, bulk environment. Detection and imaging of a single protein molecule should be possible by incorporating two-photon excitation with high sensitivity microscopy. Detection is only the first step in the study of single protein states. Fluorescence spectroscopy is required to diagnose molecular conformation. Wavelength and lifetime-resolved spectroscopy will be implemented in this project. Wavelength resolved spectra will be collected by an intensified, low noise CCD camera. Fluorescence lifetime data can be obtained by correlated single photon counting. The impact of this new methodology will be felt in many biological applications. If a single nucleotide can be detected by fluorescence, the efficiency of DNA and RNA sequencing techniques can be greatly enhanced. Further, the combination of single molecular detection and fluorescence correlation spectroscopy will allow the protein aggregation and association reactions to b e studied at dilutions down to the pico-molar level. Finally, the development of single molecule detection and spectroscopy may aid in the study of protein folding which involves a sequence of genetically programmed conformation changes. In an ensemble, the asynchronous nature of individual protein motion prevents an examination of these individual steps and only the average protein activity can be measured. However, studying one molecule at a time will resolve these individual folding steps.
