Live-Cell Superresolution Imaging by Pulsed STED Two-Photon Excitation Microscopy
Live-Cell Superresolution Imaging by Pulsed STED Two-Photon Excitation Microscopy
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DOI：
10.1016/j.bpj.2012.12.053
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发表时间：
2013-02-19
影响因子：
3.4
通讯作者：
Sabatini, Bernardo L.
中科院分区：
文献类型：
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作者：
Takasaki, Kevin T.;Ding, Jun B.;Sabatini, Bernardo L.
双光子激光扫描显微镜 (2PLSM) 允许在厚生物样品中进行荧光成像，其中吸收和散射通常会降低单光子成像方法的分辨率和信号收集。传统 2PLSM 的空间分辨率受到衍射的限制，并且 2PLSM 中用于激发的近红外波长妨碍了神经元的许多小亚细胞区室的精确成像。受激发射损耗 (STED) 显微镜是一种超分辨率成像方式，它克服了衍射带来的分辨率限制，并允许对纳米级特征进行荧光成像。在这里，我们描述了使用脉冲激发和 STED 激光器的超分辨率双光子显微镜的设计和操作。我们检查了急性组织切片中 STED 成像的深度依赖性，发现 2P 分辨率的增强范围从 20 μm 深度处的大约五倍到 90 μm 深度处的大约两倍。研究发现，分辨率的深度依赖性与耗尽效率的深度依赖性一致，表明分辨率受到 STED 激光在浑浊组织中传播的限制。最后，我们实现了 60 nm 分辨率的树突棘实时成像，并证明我们的技术可以准确量化活体脑组织中 30 μm 深的神经元形态。
Two-photon laser scanning microscopy (2PLSM) allows fluorescence imaging in thick biological samples where absorption and scattering typically degrade resolution and signal collection of one-photon imaging approaches. The spatial resolution of conventional 2PLSM is limited by diffraction, and the near-infrared wavelengths used for excitation in 2PLSM preclude the accurate imaging of many small subcellular compartments of neurons. Stimulated emission depletion (STED) microscopy is a superresolution imaging modality that overcomes the resolution limit imposed by diffraction and allows fluorescence imaging of nanoscale features. Here, we describe the design and operation of a superresolution two-photon microscope using pulsed excitation and STED lasers. We examine the depth dependence of STED imaging in acute tissue slices and find enhancement of 2P resolution ranging from approximately fivefold at 20 mu m to approximately twofold at 90-mu m deep. The depth dependence of resolution is found to be consistent with the depth dependence of depletion efficiency, suggesting resolution is limited by STED laser propagation through turbid tissue. Finally, we achieve live imaging of dendritic spines with 60-nm resolution and demonstrate that our technique allows accurate quantification of neuronal morphology up to 30-mu m deep in living brain tissue.