Many problems in contemporary molecular biology require a detailed description of molecular conformational changes.  Optical spectroscopy is a commonly used method of obtaining such structural information and has provided a wealth of knowledge about protein structure and the underlying molecular mechanics of biological processes.  Unfortunately, because they study large ensembles of molecules, spectroscopic techniques only obtain information on the average state of the molecular ensemble and have difficulty studying short lived intermediate states such as those in protein folding and motor protein function.  To address this problem we have developed near-field spectroscopy, based on the technique of near-field scanning optical microscopy (NSOM),1,2,3 for studies of protein structure and dynamics.  NSOM is a methodology for obtaining subwavelength resolution (20 - 200 nm) with the spectroscopic information afforded by conventional optical spectroscopic techniques.  The high spatial resolution of NSOM permits spectroscopic studies of individual biomolecules in high density systems such as encountered in in vivo experiments.  Unfortunately, NSOM experiments on extended samples are difficult due to its limited depth of field.  The use of two photon excitation, however, limits the excitation to a small volume near the probe, thus providing a means to suppress optical excitation of proteins far below the molecule of interest.  We have demonstrated the feasibility of this technique by exciting individual Rhodamine B molecules with 800 nm 100 fs pulsed light using an uncoated, tapered optical fiber as a probe.  The resulting images of two-photon induced fluorescence show a resolution of ~175 nm.

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NSOM Project Researchers:

Mike Lewis
Peter Wolanin

IOG Room 954
300 North Ingalls St.
Ann Arbor, MI 48109-2007