An ensemble and single-molecule fluorescence microscopy investigation of phase-separated monolayer films stained with Nile Red.

Yin Lu, Robyn Porterfield, Terri Thunder, Matthew F Paige

Index: Spectrochim. Acta. A. Mol. Biomol. Spectrosc. 78(1) , 216-23, (2011)

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Abstract

Phase-separated Langmuir-Blodgett monolayer films prepared from mixtures of arachidic acid (C19H39COOH) and perfluorotetradecanoic acid (C13F27COOH) were stained via spin-casting with the polarity sensitive phenoxazine dye Nile Red, and characterized using a combination of ensemble and single-molecule fluorescence microscopy measurements. Ensemble fluorescence microscopy and spectromicroscopy showed that Nile Red preferentially associated with the hydrogenated domains of the phase-separated films, and was strongly fluorescent in these areas of the film. These measurements, in conjunction with single-molecule fluorescence imaging experiments, also indicated that a small sub-population of dye molecules localizes on the perfluorinated regions of the sample, but that this sub-population is spectroscopically indistinguishable from that associated with the hydrogenated domains. The relative importance of selective dye adsorption and local polarity sensitivity of Nile Red for staining applications in phase-separated LB films as well as in cellular environments is discussed in context of the experimental results.Copyright © 2010 Elsevier B.V. All rights reserved.