Near-field fluorescence cross-correlation spectroscopy on planar membranes.
Near-field fluorescence cross-correlation spectroscopy on planar membranes.
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DOI：
10.1021/nn502593k
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发表时间：
2014-07-22
期刊：
影响因子：
17.1
通讯作者：
Baird, Barbara A.
中科院分区：
文献类型：
--
作者：
Kelly, Christopher V.;Wakefield, Devin L.;Holowka, David A.;Craighead, Harold G.;Baird, Barbara A.
关键词：
质膜成分在纳米尺度上的组织和动力学对于生物功能，如跨膜信号和内吞作用是必不可少的。金属薄膜中的平面化纳米孔径被证明是一种将用于多色荧光光谱的激发光限制在55±10 nm束腰的手段。该技术在平面膜上同时提供双色、亚衍射限制的荧光相关光谱和荧光互相关光谱。这项技术的制作和实施演示了模型膜和活细胞。观察到膜结合蛋白在加入多价交联剂后聚集：在支撑的脂双层上，霍乱毒素B亚单位的簇由针对该蛋白的抗体交联而形成；在活细胞上，免疫球蛋白E与其受体(FcεRI)结合在RBL肥大细胞的质膜上，在三价抗原作用下形成簇。通过荧光强度随时间的变化以及单个纳米孔径上方的时间、自相关和互相关的变化来量化膜团的形成。在实验和计算中，采用边缘为主的照明轮廓，对来自单个孔径的照明轮廓进行了分析，得到了自相关驻留时间不随孔径直径从60 nm到250 nm变化的结果。这种近场荧光互相关方法提供了对动态膜相互作用的纳米级细节的访问，并推动了近场光学方法的进一步发展。
The organization and dynamics of plasma membrane components at the nanometer scale are essential for biological functions such as transmembrane signaling and endocytosis. Planarized nanoscale apertures in a metallic film are demonstrated as a means of confining the excitation light for multicolor fluorescence spectroscopy to a 55 ± 10 nm beam waist. This technique provides simultaneous two-color, subdiffraction-limited fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy on planar membranes. The fabrication and implementation of this technique are demonstrated for both model membranes and live cells. Membrane-bound proteins were observed to cluster upon the addition of a multivalent cross-linker: On supported lipid bilayers, clusters of cholera toxin subunit B were formed upon cross-linking by an antibody specific for this protein; on living cells, immunoglobulin E bound to its receptor (FcεRI) on the plasma membranes of RBL mast cells was observed to form clusters upon exposure to a trivalent antigen. The formation of membrane clusters was quantified via fluorescence intensity vs time and changes in the temporal auto- and cross-correlations above a single nanoscale aperture. The illumination profile from a single aperture is analyzed experimentally and computationally with a rim-dominated illumination profile, yielding no change in the autocorrelation dwell time with changes in aperture diameter from 60 to 250 nm. This near-field fluorescence cross-correlation methodology provides access to nanoscale details of dynamic membrane interactions and motivates further development of near-field optical methods.
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DOI：
10.1083/jcb.200503110
发表时间：
2005-11-07
期刊：
The Journal of cell biology
影响因子：
--
作者：
Larson DR;Gosse JA;Holowka DA;Baird BA;Webb WW
通讯作者：
Webb WW
影响因子：
64.8
作者：
Ebbesen, TW;Lezec, HJ;Wolff, PA
通讯作者：
Wolff, PA
DOI：
10.1073/pnas.0511026103
发表时间：
2006-02-14
影响因子：
11.1
作者：
Gambin, Y;Lopez-Esparza, R;Urbach, W
通讯作者：
Urbach, W
影响因子：
5.4
作者：
BARSUMIAN, EL;ISERSKY, C;SIRAGANIAN, RP
通讯作者：
SIRAGANIAN, RP
影响因子：
56.9
作者：
Levene, MJ;Korlach, J;Webb, WW
通讯作者：
Webb, WW