Scanning near-field fluorescence resonance energy transfer microscopy. 1999

S A Vickery, and R C Dunn
Department of Chemistry, University of Kansas, Malott Hall, Lawrence, Kansas 66045, USA.

A new microscopic technique is demonstrated that combines attributes from both near-field scanning optical microscopy (NSOM) and fluorescence resonance energy transfer (FRET). The method relies on attaching the acceptor dye of a FRET pair to the end of a near-field fiber optic probe. Light exiting the NSOM probe, which is nonresonant with the acceptor dye, excites the donor 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 acceptor produces a red-shifted fluorescence. By monitoring this red-shifted acceptor emission, a dramatic reduction in the sample volume probed by the uncoated NSOM tip is observed. This technique is demonstrated by imaging the fluorescence from a multilayer film created using the Langmuir-Blodgett (LB) technique. The film consists of L-alpha-dipalmitoylphosphatidylcholine (DPPC) monolayers containing the donor dye, fluorescein, separated by a spacer group of three arachidic acid layers. A DPPC monolayer containing the acceptor dye, rhodamine, was also transferred onto an NSOM tip using the LB technique. 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 from the sample to the NSOM probe. This method is shown to provide enhanced depth sensitivity in fluorescence measurements, which may be particularly informative in studies on thick specimens such as cells. The technique also provides 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.

UI MeSH Term Description Entries
D008567 Membranes, Artificial Artificially produced membranes, such as semipermeable membranes used in artificial kidney dialysis (RENAL DIALYSIS), monomolecular and bimolecular membranes used as models to simulate biological CELL MEMBRANES. These membranes are also used in the process of GUIDED TISSUE REGENERATION. Artificial Membranes,Artificial Membrane,Membrane, Artificial
D008856 Microscopy, Fluorescence Microscopy of specimens stained with fluorescent dye (usually fluorescein isothiocyanate) or of naturally fluorescent materials, which emit light when exposed to ultraviolet or blue light. Immunofluorescence microscopy utilizes antibodies that are labeled with fluorescent dye. Fluorescence Microscopy,Immunofluorescence Microscopy,Microscopy, Immunofluorescence,Fluorescence Microscopies,Immunofluorescence Microscopies,Microscopies, Fluorescence,Microscopies, Immunofluorescence
D005336 Fiber Optic Technology The technology of transmitting light over long distances through strands of glass or other transparent material. Fiber Optic Technologies,Optic Technologies, Fiber,Optic Technology, Fiber,Technologies, Fiber Optic,Technology, Fiber Optic
D005456 Fluorescent Dyes Chemicals that emit light after excitation by light. The wave length of the emitted light is usually longer than that of the incident light. Fluorochromes are substances that cause fluorescence in other substances, i.e., dyes used to mark or label other compounds with fluorescent tags. Flourescent Agent,Fluorescent Dye,Fluorescent Probe,Fluorescent Probes,Fluorochrome,Fluorochromes,Fluorogenic Substrates,Fluorescence Agents,Fluorescent Agents,Fluorogenic Substrate,Agents, Fluorescence,Agents, Fluorescent,Dyes, Fluorescent,Probes, Fluorescent,Substrates, Fluorogenic
D001703 Biophysics The study of PHYSICAL PHENOMENA and PHYSICAL PROCESSES as applied to living things. Mechanobiology
D012235 Rhodamines A family of 3,6-di(substituted-amino)-9-benzoate derivatives of xanthene that are used as dyes and as indicators for various metals; also used as fluorescent tracers in histochemistry. Rhodamine
D013050 Spectrometry, Fluorescence Measurement of the intensity and quality of fluorescence. Fluorescence Spectrophotometry,Fluorescence Spectroscopy,Spectrofluorometry,Fluorescence Spectrometry,Spectrophotometry, Fluorescence,Spectroscopy, Fluorescence
D015060 1,2-Dipalmitoylphosphatidylcholine Synthetic phospholipid used in liposomes and lipid bilayers to study biological membranes. It is also a major constituent of PULMONARY SURFACTANTS. Dipalmitoyllecithin,1,2-Dihexadecyl-sn-Glycerophosphocholine,1,2-Dipalmitoyl-Glycerophosphocholine,Dipalmitoyl Phosphatidylcholine,Dipalmitoylglycerophosphocholine,Dipalmitoylphosphatidylcholine,1,2 Dihexadecyl sn Glycerophosphocholine,1,2 Dipalmitoyl Glycerophosphocholine,1,2 Dipalmitoylphosphatidylcholine,Phosphatidylcholine, Dipalmitoyl
D055100 Optical Fibers Thin strands of transparent material, usually glass, that are used for transmitting light waves over long distances. Fiber Optic Cables,Cable, Fiber Optic,Cables, Fiber Optic,Fiber Optic Cable,Fiber, Optical,Fibers, Optical,Optic Cable, Fiber,Optic Cables, Fiber,Optical Fiber
D055592 Biophysical Phenomena The physical characteristics and processes of biological systems. Biophysical Concepts,Biophysical Processes,Biophysical Phenomenon,Biophysical Process,Biophysical Concept,Concept, Biophysical,Concepts, Biophysical,Phenomena, Biophysical,Phenomenon, Biophysical,Process, Biophysical,Processes, Biophysical

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