Interdisciplinary Institute for Neuroscience, Université de Bordeaux, Bordeaux, France.
Biophys J. 2013 Feb 19;104(4):778-85. doi: 10.1016/j.bpj.2012.12.054.
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 ~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.
许多细胞结构和细胞器太小，无法通过传统的光学显微镜进行适当的解析。这对于树突棘和神经胶质突起尤其如此，它们非常小，具有动态性，并且嵌入在密集的组织中，使得在实际实验条件下对其进行成像变得非常困难。双光子显微镜是目前在厚的活体组织切片中进行成像的首选方法，无论是在急性脑切片还是在体中都是如此。然而，双光子显微镜的空间分辨率受到光的衍射限制，约为 350nm，不足以解析神经形态的许多重要细节，例如棘突颈部的宽度或薄的神经胶质突起。最近开发的超分辨率方法，如受激发射损耗显微镜，为活体组织成像设定了新的光学分辨率标准。然而，在急性脑切片中尚未实现具有显著亚衍射分辨率的超分辨率成像的重要目标。为了克服这一限制，我们开发了一种基于双光子激发和脉冲受激发射损耗显微镜的新型显微镜，该显微镜在使用长工作距离物镜的急性脑切片中提供了前所未有的空间分辨率和出色的实验访问权限。新型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，并且与活细胞中的时移和双色超分辨率成像兼容。我们通过在急性脑切片的表面以下对树突棘和小神经胶质细胞的形态进行成像，证明了这种纳米显微镜方法在脑片生理学中的潜力。
