Nanoscale protein diffusion by STED-based pair correlation analysis.
Nanoscale protein diffusion by STED-based pair correlation analysis.
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DOI：
10.1371/journal.pone.0099619
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Bizzarri R
中科院分区：
文献类型：
--
作者：
Bianchini P;Cardarelli F;Di Luca M;Diaspro A;Bizzarri R
我们首次描述了交叉对相关函数分析（对相关分析或pCF）和受激发射耗尽（STED）之间的组合，以获得空间分辨率低于光学衍射极限（超分辨率）的扩散图。我们的方法在以高和低信噪比为特征的系统中进行了测试，即分别在活的中国人卵巢细胞中瞬时表达的携带几种（>100）活性荧光蛋白和单体荧光蛋白的衣壳样颗粒（CLP）。后一种系统代表了荧光蛋白嵌合体活细胞研究中遇到的常见条件。STED-pCF的空间分辨率被认为是约110 nm，与传统的共焦采集的两倍以上的改善。我们成功地应用了我们的方法来突出如何接近核膜影响蛋白质的流动性特征，积极进口到细胞核中的活细胞。值得注意的是，STED-pCF揭示了扩散的局部障碍的存在，以及在距离核膜两侧高达500-700 nm处存在缓慢组分。该组分的迁移率类似于先前描述的转运复合物的迁移率。值得注意的是，所有这些特征在传统的共焦模式下都是不可见的。
We describe for the first time the combination between cross-pair correlation function analysis (pair correlation analysis or pCF) and stimulated emission depletion (STED) to obtain diffusion maps at spatial resolution below the optical diffraction limit (super-resolution). Our approach was tested in systems characterized by high and low signal to noise ratio, i.e. Capsid Like Particles (CLPs) bearing several (>100) active fluorescent proteins and monomeric fluorescent proteins transiently expressed in living Chinese Hamster Ovary cells, respectively. The latter system represents the usual condition encountered in living cell studies on fluorescent protein chimeras. Spatial resolution of STED-pCF was found to be about 110 nm, with a more than twofold improvement over conventional confocal acquisition. We successfully applied our method to highlight how the proximity to nuclear envelope affects the mobility features of proteins actively imported into the nucleus in living cells. Remarkably, STED-pCF unveiled the existence of local barriers to diffusion as well as the presence of a slow component at distances up to 500–700 nm from either sides of nuclear envelope. The mobility of this component is similar to that previously described for transport complexes. Remarkably, all these features were invisible in conventional confocal mode.
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影响因子：
3.8
作者：
Moneron, Gael;Medda, Rebecca;Hell, Stefan W.
通讯作者：
Hell, Stefan W.
DOI：
10.1083/jcb.201004104
发表时间：
2010-05-31
期刊：
The Journal of cell biology
影响因子：
--
作者：
Linkert M;Rueden CT;Allan C;Burel JM;Moore W;Patterson A;Loranger B;Moore J;Neves C;Macdonald D;Tarkowska A;Sticco C;Hill E;Rossner M;Eliceiri KW;Swedlow JR
通讯作者：
Swedlow JR
DOI：
10.1073/pnas.1006731107
发表时间：
2010-09-21
影响因子：
11.1
作者：
Hinde, Elizabeth;Cardarelli, Francesco;Gratton, Enrico
通讯作者：
Gratton, Enrico
影响因子：
3.8
作者：
Leutenegger, Marcel;Eggeling, Christian;Hell, Stefan W.
通讯作者：
Hell, Stefan W.
影响因子：
3.4
作者：
Digman, Michelle A.;Brown, Claire M.;Gratton, Enrico
通讯作者：
Gratton, Enrico