Two photon fluorescence microscopy of coexisting lipid domains in giant unilamellar vesicles of binary phospholipid mixtures
Two photon fluorescence microscopy of coexisting lipid domains in giant unilamellar vesicles of binary phospholipid mixtures
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DOI：
10.1016/s0006-3495(00)76592-1
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发表时间：
2000-01-01
影响因子：
3.4
通讯作者：
Gratton, E
中科院分区：
文献类型：
--
作者：
Bagatolli, LA;Gratton, E
利用双光子激发荧光显微镜的切片能力，获得了不同磷脂混合物（1，2-二棕榈酰-sn-甘油-3-磷酸胆碱/1，2-二月桂酰-sn-甘油-3-磷酸胆碱（DPPC/DLPC）1：1（mol/mol）和1，2-二棕榈酰-sn-甘油-3-磷酸乙醇胺/1，2-二棕榈酰-sn-甘油-3-磷酸胆碱（DPPE/DPPC）7：3和3：7（mol/mol））在不同温度下形成的巨大单层囊泡（GUV）的图像。6-十二烷酰基-2-二甲氨基-萘（LAURDAN）、6-丙酰基-2-二甲氨基-萘（PRODAN）和丽丝胺罗丹明B 1，2-二十六烷酰基-sn-甘油基-3-磷酸乙醇胺（N-Rh-DPPE）用作荧光探针以揭示GUV中的结构域共存。Mle报道了无支撑脂质双层中脂质结构域形态的首次表征。从LAURDAN强度图像的激发广义偏振函数（GP）计算在不同的温度下的相态的脂质结构域的特征。在每种脂质混合物的相图的基础上，我们发现在对应于所有脂质混合物中的流体区域的温度下，在GUV图像中具有均匀的荧光分布。在对应于相共存区域的温度下，我们观察到不同大小和形状的脂质结构域，这取决于脂质样品的组成。在由DPPE/DPPC混合物形成的GUV的情况下，凝胶DPPE域呈现不同的形状，例如六边形、菱形、六角星星、哑铃形或树枝形。在相共存区，凝胶DPPE微区随着温度的降低而移动和增长。分离的域保持在GUV在对应于固体区域的温度下，显示出固-固不相容性。在由DLPC/DPPC 1：1（mol/mol）混合物组成的GUV中发现了不同的形态。在对应于相共存的温度下，我们观察到凝胶域作为线缺陷在GUV表面。随着温度的降低，这些线移动并变得更粗。通过LAURDAN GP直方图判断，我们得出结论，DPPE/DPPC和DLPC/DPPC混合物在相共存区的脂相特征不同。在DPPE/DPPC混合物中，纯凝胶和纯液体区域之间共存，而在DLPC/DPPC 1：1（mol/mol）混合物中，我们观察到一个相对另一个相的强烈影响。在所有情况下，域跨越膜的内部和外部小叶，表明脂质膜的内部和外部单层之间的强耦合。这种观察对于无支撑的脂质双层也是新颖的。
Images of giant unilamellar vesicles (GUVs) formed by different phospholipid mixtures (1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dilauroyl-sn-glycero-3-phosphocholine (DPPC/DLPC) 1:1 (mol/mol), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine/1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPE/DPPC), 7:3 and 3:7 (mol/mol) at different temperatures were obtained by exploiting the sectioning capability of a two-photon excitation fluorescence microscope. 6-Dodecanoyl-2-dimethylamino-naphthalene (LAURDAN), 6-propionyl-2-dimethylamino-naphthalene (PRODAN), and Lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (N-Rh-DPPE) were used as fluorescent probes to reveal domain coexistence in the GUVs. Mle report the first characterization of the morphology of lipid domains in unsupported lipid bilayers. From the LAURDAN intensity images the excitation generalized polarization function (GP) was calculated at different temperatures to characterize the phase state of the lipid domain. On the basis of the phase diagram of each lipid mixture, we found a homogeneous fluorescence distribution in the GUV images at temperatures corresponding to the fluid region in all lipid mixtures. At temperatures corresponding to the phase coexistence region we observed lipid domains of different sizes and shapes, depending on the lipid sample composition. In the case of GUVs formed by DPPE/DPPC mixture, the gel DPPE domains present different shapes, such as hexagonal, rhombic, six-cornered star, dumbbell, or dendritic. At the phase coexistence region, the gel DPPE domains are moving and growing as the temperature decreases. Separated domains remain in the GUVs at temperatures corresponding to the solid region, showing solid-solid immiscibility. A different morphology was found in GUVs composed of DLPC/DPPC 1:1 (mol/mol) mixtures. At temperatures corresponding to the phase coexistence, we observed the gel domains as line defects in the GUV surface. These lines move and become thicker as the temperature decreases. As judged by the LAURDAN GP histogram, we concluded that the lipid phase characteristics at the phase coexistence region are different between the DPPE/DPPC and DLPC/DPPC mixtures. In the DPPE/DPPC mixture the coexistence is between pure gel and pure liquid domains, while in the DLPC/DPPC 1:1 (mol/mol) mixture we observed a strong influence of one phase on the other. In all cases the domains span the inner and outer leaflets of the membrane, suggesting a strong coupling between the inner and outer monolayers of the lipid membrane. This observation is also novel for unsupported lipid bilayers.