Two-Photon Excitation STED Microscopy in Two Colors in Acute Brain Slices
Two-Photon Excitation STED Microscopy in Two Colors in Acute Brain Slices
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DOI：
10.1016/j.bpj.2012.12.054
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发表时间：
2013-02-19
影响因子：
3.4
通讯作者：
Naegerl, U. Valentin
中科院分区：
文献类型：
--
作者：
Bethge, Philipp;Chereau, Ronan;Naegerl, U. Valentin
许多细胞结构和细胞器太小，无法通过常规光学显微镜正确分辨。这对于树突棘和神经胶质过程尤其如此，它们非常小，动态，并且嵌入致密组织中，使得难以在现实的实验条件下对其进行成像。双光子显微镜是目前在厚活组织制备成像的首选方法，无论是在急性脑切片和体内。然而，由于光的衍射，双光子显微镜的空间分辨率被限制在350 nm左右，不足以分辨神经形态的许多重要细节，例如脊柱颈的宽度或薄的神经胶质过程。最近开发的超分辨率方法，如受激发射损耗显微镜，已经设定了新的标准的光学分辨率成像活组织。然而，具有显著亚衍射分辨率的超分辨率成像的重要目标尚未在急性脑切片中实现。为了克服这一限制，我们已经开发出一种新的显微镜的基础上双光子激发和脉冲受激发射损耗显微镜，它提供了前所未有的空间分辨率和优秀的实验访问急性脑切片使用长工作距离的目标。新的显微镜将常规双光子显微镜的空间分辨率提高了四到六倍，并且与活细胞中的延时和同时双色超分辨率成像兼容。我们通过对急性脑切片表面以下的树突棘和小胶质细胞的形态成像，证明了这种纳米显微镜方法用于脑切片生理学的潜力。
Many cellular structures and organelles are too small to be properly resolved by conventional light microscopy. This is particularly true for dendritic spines and glial processes, which are very small, dynamic, and embedded in dense tissue, making it difficult to image them under realistic experimental conditions. Two-photon microscopy is currently the method of choice for imaging in thick living tissue preparations, both in acute brain slices and in vivo. However, the spatial resolution of a two-photon microscope, which is limited to similar to 350 nm by the diffraction of light, is not sufficient for resolving many important details of neural morphology, such as the width of spine necks or thin glial processes. Recently developed superresolution approaches, such as stimulated emission depletion microscopy, have set new standards of optical resolution in imaging living tissue. However, the important goal of superresolution imaging with significant subdiffraction resolution has not yet been accomplished in acute brain slices. To overcome this limitation, we have developed a new microscope based on two-photon excitation and pulsed stimulated emission depletion microscopy, which provides unprecedented spatial resolution and excellent experimental access in acute brain slices using a long-working distance objective. The new microscope improves on the spatial resolution of a regular two-photon microscope by a factor of four to six, and it is compatible with time-lapse and simultaneous two-color superresolution imaging in living cells. We demonstrate the potential of this nanoscopy approach for brain slice physiology by imaging the morphology of dendritic spines and microglial cells well below the surface of acute brain slices.