Characterization of lipid bilayer phases by confocal microscopy and fluorescence correlation spectroscopy
Characterization of lipid bilayer phases by confocal microscopy and fluorescence correlation spectroscopy
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DOI：
10.1073/pnas.96.15.8461
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发表时间：
1999-07-20
影响因子：
11.1
通讯作者：
Feigenson, GW
中科院分区：
文献类型：
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作者：
Korlach, J;Schwille, P;Feigenson, GW
我们报告了应用共聚焦成像和荧光相关光谱(FCS)来表征用于生物膜的化学定义良好的脂质双层模型。用1，1‘-dieicosanyl-3，3，3’，3‘-tetramethylindocarbocyanine高氯酸盐(DiI-C-20)和2-(4，4-difluoro-5，7-dimethyl-4-bora-3a，4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine(BODIPY-PC)两种荧光探针，在共聚焦荧光显微镜下观察了双月桂酰磷脂酰胆碱/二棕榈酰磷脂酰胆碱/胆固醇的巨大单层囊泡的相分离情况。共聚焦Z扫描通过巨大的单层囊泡的三维图像重建揭示了这些囊泡表面以全二维分辨率共存的相区的各向异性形态。这种方法通过直观的可视化演示了类似相区在相对的单分子层中的准确叠加，从而回答了一个长期悬而未决的问题。研究发现，胆固醇会导致共存相区的相界形状发生显著变化。为了进一步表征这些物相，用BCS测量了DII-C-20的平移扩散系数D-T。在接近25℃的温度下，流体相的DT值接近3×10(-8)cm(2)/S，高胆固醇相的DT值接近2×10(-9)cm(2)/S，与流体相共存的空间有序相的DT值接近2×10(-10)cm(2)/S，在有利的情况下，FCS可以区分单个囊泡上两相共存区域的两个不同的DT值。
We report the application of confocal imaging and fluorescence correlation spectroscopy (FCS) to characterize chemically well-defined lipid bilayer models for biomembranes. Giant unilamellar vesicles of dilauroyl phosphatidylcholine/dipalmitoyl phosphatidylcholine (DLPC/ DPPC)/cholesterol were imaged by confocal fluorescence microscopy with two fluorescent probes, 1,1'-dieicosanyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI-C-20) and 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (Bodipy-PC), Phase separation was visualized by differential probe partition into the coexisting phases. Three-dimensional image reconstructions of confocal z-scans through giant unilamellar vesicles reveal the anisotropic morphology of coexisting phase domains on the surface of these vesicles with full two-dimensional resolution. This method demonstrates by direct visualization the exact superposition of like phase domains in apposing monolayers, thus answering a long-standing open question. Cholesterol was found to induce a marked change in the phase boundary shapes of the coexisting phase domains. To further characterize the phases, the translational diffusion coefficient, D-T, of the DiI-C-20 was measured by BCS. DT values at similar to 25 degrees C ranged from similar to 3 x 10(-8) cm(2)/s in the fluid phase, to similar to 2 x 10(-9) cm(2)/s in high-cholesterol-content phases, to similar to 2 x 10(-10) cm(2)/s in the spatially ordered phases that coexist with fluid phases, In favorable cases, FCS could distinguish two different values of D-T in a region of two-phase coexistence on a single vesicle.