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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Mike Lewis Peter Wolanin