Fluorescent Dimers of Merocyanine 540 (MC540) in the Gel Phase of Phosphatidylcholine Liposomes

Abstract

Abstract— Fluorescence emission from merocyanine 540 (MC540) dimers was observed in dipalmitoylphosphatidylcholine (DPPC) vesicles. This unusual behavior was observed only for vesicles in the gel‐phase state. No dimer fluorescence was observed either in monopalmitoylphosphati‐dylcholine (C16PC) micelles or in liquid‐crystalline DPPC vesicles, indicating that dimer fluorescence efficiency increases in highly packed interfaces. The excitonic theory of Kasha was used to interpret the spectral features. The overall fluorescence quantum yield (φr) decreases with decreasing lipid: probe ratio, not only because of the presence of a weakly fluorescent dimer that absorbs a high fraction of the total absorbed light but also due to quenching of monomer emission. This suggests the existence of probe domains. The dimer fluorescence quantum yields (φm) were estimated in DPPC large unilamellar vesicles (LUV) and DPPC multilamellar vesicles. The dependence of φr with probe concentration is compatible with values of φm lower than 0.05. The dimerization equilibrium of MC540 in C16PC micelles and DPPC‐LUV was also studied. Apparent dimerization equilibrium constants, Kdapp and dimer absorption spectrum were calculated in C16PC micelles for the first time. The dimerization equilibrium constant in DPPC‐LUV was calculated and discussed in terms of the fraction of volume occupied by the lipid phase.

Combined use of steady-state fluorescence emission and anisotropy of merocyanine 540 to distinguish crystalline, gel, ripple, and liquid crystalline phases in dipalmitoylphosphatidylcholine bilayers

“…[3,4,8-12]). Because of the sensitivity of MC540 to lipid-packing density and the ability of the probe to partition into different membrane locations depending on lipid phase [7,13-17], we postulated that it might be able to provide detailed information about membrane dynamics through multiple simultaneous steady-state measurements. We have used bilayers made of dipalmitoylphosphatidylcholine (DPPC) with and without cholesterol to assess the extent to which such might be the case.…”

“…The monomeric form of MC540 resides near the lipid head groups at an orientation parallel to the phospholipid chains [16]. Alternatively, MC540 can form an anti-parallel "sandwich" dimer [17]. It has been proposed that this dimer occupies a different region of the membrane compared to the monomer, although the exact orientation of the probe and whether that region is deep or superficial in the membrane are uncertain [16,37].…”

“…It has been proposed that this dimer occupies a different region of the membrane compared to the monomer, although the exact orientation of the probe and whether that region is deep or superficial in the membrane are uncertain [16,37]. Emission intensity spectra show that the monomer fluoresces at a shorter wavelength than the dimer (585 nm and 621 nm, respectively) [17,37]. While the overall dimer fluorescence intensity is lower than that of monomers, dimers do contribute a noticeable amount to the emission intensity of MC540 when they reside in a highly packed environment such as exists in the membrane below the main phase transition [17].…”

Super-resolution Microscopy of Lipid Bilayer Phases

“…The dimer form is prevalent in most lipids but the equilibrium is sensitive to the lipid packing order. The apparent dimerization equilibrium constants (ratios of the dimer concentration to the square of the monomer concentration) in phosphatidylcholine (PC) suspensions are in the range of 10 3 to 10 6 M −1 , depending on the lipid packing, temperature, and [lipid]/[MC 540] ratio 31, 32, 37, 38 . The kinetics leading to the attainment of the monomer-dimer equilibrium have been studied by several research groups 28, 31, 32, 36, 39 .…”

“…The fluorescence yield of monomeric MC540 is reported as 0.6 in DMPC liposomes and micelles while in water it is 0.0527. Dimeric MC 540 in liposomes absorbs at 540 nm and emits with low yield at 625 nm 25, 38 . In the present work, fluorescence spectra were recorded for DOPC SLB and DPPC SLB containing MC 540.…”