Superresolution Imaging of Multiple Fluorescent Proteins with Highly Overlapping Emission Spectra in Living Cells
Superresolution Imaging of Multiple Fluorescent Proteins with Highly Overlapping Emission Spectra in Living Cells
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DOI：
10.1016/j.bpj.2011.07.049
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发表时间：
2011-09-21
影响因子：
3.4
通讯作者：
Hess, Samuel T.
中科院分区：
文献类型：
--
作者：
Gunewardene, Mudalige S.;Subach, Fedor V.;Hess, Samuel T.
基于定位的超分辨率光学成像正在迅速普及，但具有不同发射光谱的遗传编码的光活化荧光探针的有限可用性阻碍了活细胞中多个分子物种的同时可视化。我们介绍PAmKate，单体光活化远红荧光蛋白，这有利于同时成像的三个光活化蛋白在哺乳动物细胞中使用荧光光活化定位显微镜（FPALM）。成功的探针识别是通过测量两个不同的光谱通道中的荧光发射强度，跨越仅类似于100 nm的可见光谱。筏，非筏，和cytokerion相关的蛋白质同时成像在活的和固定的成纤维细胞共表达Dendra 2-血凝素，PAmKate转铁蛋白受体，PAmCherry 1-β-肌动蛋白融合结构，揭示膜蛋白和膜相关的肌动蛋白结构之间的相关性。
Localization-based superresolution optical imaging is rapidly gaining popularity, yet limited availability of genetically encoded photoactivatable fluorescent probes with distinct emission spectra impedes simultaneous visualization of multiple molecular species in living cells. We introduce PAmKate, a monomeric photoactivatable far-red fluorescent protein, which facilitates simultaneous imaging of three photoactivatable proteins in mammalian cells using fluorescence photoactivation localization microscopy (FPALM). Successful probe identification was achieved by measuring the fluorescence emission intensity in two distinct spectral channels spanning only similar to 100 nm of the visible spectrum. Raft-, non-raft-, and cytoskeleton-associated proteins were simultaneously imaged in both live and fixed fibroblasts coexpressing Dendra2-hemagglutinin, PAmKate-transferrin receptor, and PAmCherry1-beta-actin fusion constructs, revealing correlations between the membrane proteins and membrane-associated actin structures.