Combined Use of Steady-State Fluorescence Emission and Anisotropy of Merocyanine 540 To Distinguish Crystalline, Gel, Ripple, and Liquid Crystalline Phases in Dipalmitoylphosphatidylcholine Bilayers

Abstract

The various lamellar phases of dipalmitoylphosphadtidylcholine bilayers with and without cholesterol were used to assess the versatility of the fluorescent probe merocyanine 540 through simultaneous measurements of emission intensity, spectral shape, and steady-state anisotropy. Induction of the crystalline phase (L c ') by preincubation at 4°C produced a wavelength dependence of anisotropy which was strong at 15 and 25°C, weak at 38°C, and minimal above the main transition (>~41.5°C) or after returning the temperature from 46 to 25°C. The profile of anisotropy values across this temperature range revealed the ability of the probe to detect crystalline, gel (L β '), and liquid crystalline (L α ) phases. The temperature dependence of fluorescence intensity was additionally able to distinguish between the ripple (P β ') and gel phases. In contrast, the shape of the emission spectrum, quantified as the ratio of merocyanine monomer and dimer peaks (585 and 621 nm), was primarily sensitive to the crystalline and gel phases because dimer fluorescence requires a highly-ordered environment. This requirement also explained the diminution of anisotropy wavelength dependence above 25°C. Repetition of experiments with vesicles containing cholesterol allowed creation of a phase map. Superimposition of data from the three simultaneous measurements provided details about the various phase regions in the map not discernible from any one of the three alone. The results were applied to assessment of calcium-induced membrane changes in living cells.