Scanning near-field fluorescence resonance energy transfer microscopy

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.