Supraresolution imaging in brain slices using stimulated-emission depletion two-photon laser scanning microscopy.
Supraresolution imaging in brain slices using stimulated-emission depletion two-photon laser scanning microscopy.
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DOI：
10.1016/j.neuron.2009.07.011
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发表时间：
2009-08-27
期刊：
影响因子：
16.2
通讯作者：
Sabatini, Bernardo L.
中科院分区：
文献类型：
--
作者：
Ding, Jun B.;Takasaki, Kevin T.;Sabatini, Bernardo L.
双光子激光扫描显微镜（2 PLSM）允许前所未有的荧光成像神经组织内的神经元结构和功能。然而，这种方法的分辨率是穷人相比，传统的共聚焦显微镜。在这里，我们展示了脑切片内的超分辨率2 PLSM。超过衍射极限的成像是通过使用近红外（NIR）激光器进行脉冲双光子激发和连续波刺激发射耗尽（STED）来实现的。此外，我们证明了Alexa Fluor-594，一种常用于活细胞和固定组织荧光成像的明亮荧光团，适用于STED 2 PLSM。STED 2 PLSM超分辨率显微镜在径向方向上的分辨率比传统2 PLSM提高了约3倍，揭示了位于脑切片表面以下约100微米处的树突棘结构的更多细节。理论上可以进一步提高分辨率，这表明STED 2 PLSM将允许位于相对完整脑组织中的神经元结构的纳米级成像。
Two-photon laser scanning microscopy (2PLSM) has allowed unprecedented fluorescent imaging of neuronal structure and function within neural tissue. However, the resolution of this approach is poor compared to that of conventional confocal microscopy. Here we demonstrate supraresolution 2PLSM within brain slices. Imaging beyond the diffraction limit is accomplished by using near-infrared (NIR) lasers for both pulsed 2-photon excitation and continuous wave stimulation emission depletion (STED). Furthermore, we demonstrate that Alexa Fluor-594, a bright fluorophore commonly used for both live cell and fixed tissue fluorescence imaging, is suitable for STED 2PLSM. STED 2PLSM supraresolution microscopy achieves approximately 3 fold improvement in resolution in the radial direction over conventional 2PLSM, revealing greater detail in the structure of dendritic spines located ~100 microns below the surface of brain slices. Further improvements in resolution are theoretically achievable, suggesting that STED 2PLSM will permit nanoscale imaging of neuronal structures located in relatively intact brain tissue.
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