Three-Dimensional Super-Resolution in Eukaryotic Cells Using the Double-Helix Point Spread Function.
Three-Dimensional Super-Resolution in Eukaryotic Cells Using the Double-Helix Point Spread Function.
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DOI：
10.1016/j.bpj.2017.02.023
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发表时间：
2017-04-11
影响因子：
3.4
通讯作者：
Lee SF
中科院分区：
文献类型：
--
作者：
Carr AR;Ponjavic A;Basu S;McColl J;Santos AM;Davis S;Laue ED;Klenerman D;Lee SF
单分子定位显微镜，通常是基于全内反射照明，已经采取了我们的理解蛋白质组织和细胞动力学超出衍射极限。然而，生物系统存在于一个复杂的三维环境中，这就需要开发新的技术，包括双螺旋点扩散函数（DHPSF），以准确地可视化生物过程。到目前为止，DHPSF方法的应用仅限于研究相对较小的原核细胞。通过匹配的折射率的物镜透镜浸没液体的样品介质，我们展示了DHPSF成像高达15 μ m厚的整个真核细胞体积在三到五个成像平面。我们通过探索受体触发后人类T细胞中的大规模膜重组，以及通过使用单粒子跟踪来成像几种哺乳动物蛋白质，包括T细胞和胚胎干细胞中的膜、细胞质和核蛋白，来说明DHPSF的能力。
Single-molecule localization microscopy, typically based on total internal reflection illumination, has taken our understanding of protein organization and dynamics in cells beyond the diffraction limit. However, biological systems exist in a complicated three-dimensional environment, which has required the development of new techniques, including the double-helix point spread function (DHPSF), to accurately visualize biological processes. The application of the DHPSF approach has so far been limited to the study of relatively small prokaryotic cells. By matching the refractive index of the objective lens immersion liquid to that of the sample media, we demonstrate DHPSF imaging of up to 15-μm-thick whole eukaryotic cell volumes in three to five imaging planes. We illustrate the capabilities of the DHPSF by exploring large-scale membrane reorganization in human T cells after receptor triggering, and by using single-particle tracking to image several mammalian proteins, including membrane, cytoplasmic, and nuclear proteins in T cells and embryonic stem cells.