Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Biophys J. 2011 Sep 7;101(5):1277-84. doi: 10.1016/j.bpj.2011.07.027.
It is difficult to investigate the mechanisms that mediate long-term changes in synapse function because synapses are small and deeply embedded inside brain tissue. Although recent fluorescence nanoscopy techniques afford improved resolution, they have so far been restricted to dissociated cells or tissue surfaces. However, to study synapses under realistic conditions, one must image several cell layers deep inside more-intact, three-dimensional preparations that exhibit strong light scattering, such as brain slices or brains in vivo. Using aberration-reducing optics, we demonstrate that it is possible to achieve stimulated emission depletion superresolution imaging deep inside scattering biological tissue. To illustrate the power of this novel (to our knowledge) approach, we resolved distinct distributions of actin inside dendrites and spines with a resolution of 60-80 nm in living organotypic brain slices at depths up to 120 μm. In addition, time-lapse stimulated emission depletion imaging revealed changes in actin-based structures inside spines and spine necks, and showed that these dynamics can be modulated by neuronal activity. Our approach greatly facilitates investigations of actin dynamics at the nanoscale within functionally intact brain tissue.
由于突触体积小且深埋在脑组织内部，因此很难研究介导突触功能长期变化的机制。尽管最近的荧光纳米显微镜技术提供了更高的分辨率，但它们迄今为止仅限于分离细胞或组织表面。然而，要在更真实的条件下研究突触，必须对具有强光散射的更完整的三维制剂（如脑切片或活体脑）进行多层成像。我们使用像差校正光学元件，证明了在散射生物组织内部实现受激发射耗散超分辨率成像的可能性。为了说明这种新颖的（据我们所知）方法的强大功能，我们在深度达 120μm 的活体器官型脑切片中以 60-80nm 的分辨率解析了树突和棘突内的肌动蛋白的不同分布。此外，延时受激发射耗散成像揭示了棘突和棘突颈部内基于肌动蛋白的结构的变化，并表明神经元活动可以调节这些动力学。我们的方法极大地促进了在功能完整的脑组织内对纳米尺度上肌动蛋白动力学的研究。
