A new microscopic technique is demonstrated that combines attributes from b
oth near-field scanning optical microscopy (NSOM) and fluorescence resonanc
e energy transfer (FRET). The method relies on attaching the acceptor dye o
f a FRET pair to the end of a near-field fiber optic probe. Light exiting t
he NSOM probe, which is nonresonant with the acceptor dye, excites the dono
r dye introduced into a sample. As the tip approaches the sample containing
the donor dye, energy transfer from the excited donor to the tip-bound acc
eptor produces a red-shifted fluorescence. By monitoring this red-shifted a
cceptor emission, a dramatic reduction in the sample volume probed by the u
ncoated NSOM tip is observed. This technique is demonstrated by imaging the
fluorescence from a multilayer film created using the Langmuir-Blodgett (L
B) technique. The film consists of L-alpha-dipalmitoylphosphatidylcholine (
DPPC) monolayers containing the donor dye, fluorescein, separated by a spac
er group of three arachidic acid layers. A DPPC monolayer containing the ac
ceptor dye, rhodamine, was also transferred onto an NSOM tip using the LB t
echnique. Using this modified probe, fluorescence images of the multilayer
film reveal distinct differences between images collected monitoring either
the donor or acceptor emission. The latter results from energy transfer fr
om the sample to the NSOM probe. This method is shown to provide enhanced d
epth sensitivity in fluorescence measurements, which may be particularly in
formative in studies On thick specimens such as cells. The technique also p
rovides a mechanism for obtaining high spatial resolution without the need
for a metal coating around the NSOM probe and Should work equally well with
nonwaveguide probes such as atomic force microscopy tips. This may lead to
dramatically improved spatial resolution in fluorescence imaging.