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 (Lc') by pre-incubation 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 (Lβ'), and liquid crystalline (Lα) phases. The temperature dependence of fluorescence intensity was additionally able to distinguish between the ripple (Pβ') 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.PACS Codes: 87.16.dt
Highlights
Fluorescence spectroscopy is a useful biological technique for studying membrane structure that can be applied directly to living cells
We have examined the versatility of using simultaneous measurements of Merocyanine 540 (MC540) anisotropy, emission intensity, and spectral shape to resolve the various DPPC lamellar phases
The wavelength dependence persisted, the overall anisotropy values were lower compared to those at 15°C (slope 95% confidence interval = -0.005 to -0.004 nm-1, p
Summary
Fluorescence spectroscopy is a useful biological technique for studying membrane structure that can be applied directly to living cells. If one could increase the amount of biophysical information available from those measurements, it would reduce the need for dual labeling or comparisons of parallel experiments with different probes It has been found with the probe laurdan that measurement of both steadystate anisotropy and emission spectrum shape can yield more detailed information about membrane phase properties in both artificial and natural membranes than either measurement alone [1,2]. Its emission intensity is environment-sensitive, which allows it to be used in flow cytometry experiments to quantify cell subpopulations with differing biophysical membrane properties [3,4]. It binds to the plasma membrane of most cells at low concentrations We have used bilayers made of dipalmitoylphosphatidylcholine (DPPC) with and without cholesterol to assess the extent to which such might be the case
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