Scanning Near-Field Fluorescence Resonance Energy Transfer Microscopy
| Autor: | Robert C. Dunn, Sarah A. Vickery |
|---|---|
| Rok vydání: | 1999 |
| Předmět: |
Fluorescence-lifetime imaging microscopy
1 2-Dipalmitoylphosphatidylcholine Biophysics Analytical chemistry 02 engineering and technology 7. Clean energy Biophysical Phenomena law.invention Rhodamine 03 medical and health sciences chemistry.chemical_compound Optical microscope law Microscopy Fiber Optic Technology Optical Fibers Fluorescent Dyes 030304 developmental biology 0303 health sciences Rhodamines Chemistry Membranes Artificial 021001 nanoscience & nanotechnology Fluorescence Acceptor Spectrometry Fluorescence Förster resonance energy transfer Microscopy Fluorescence Fluorescein Near-field scanning optical microscope 0210 nano-technology Research Article |
| Zdroj: | Biophysical Journal. 76:1812-1818 |
| ISSN: | 0006-3495 |
| DOI: | 10.1016/s0006-3495(99)77341-8 |
| Popis: | 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-α-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. |
| Databáze: | OpenAIRE |
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