Wavefront-Engineering Microscopy Group, Neurophysiology and New Microscopies Laboratory, Centre National de la Recherche Scientifique UMR8154, Institut National de la Santé et de la Recherche Médicale U603, Université Paris Descartes, 45 Rue des Saints Pères, F-75006 Paris, France.
Proc Natl Acad Sci U S A. 2011 Dec 6;108(49):19504-9. doi: 10.1073/pnas.1109111108. Epub 2011 Nov 9.
Access to three-dimensional structures in the brain is fundamental to probe signal processing at multiple levels, from integration of synaptic inputs to network activity mapping. Here, we present an optical method for independent three-dimensional photoactivation and imaging by combination of digital holography with remote-focusing. We experimentally demonstrate compensation of spherical aberration for out-of-focus imaging in a range of at least 300 μm, as well as scanless imaging along oblique planes. We apply this method to perform functional imaging along tilted dendrites of hippocampal pyramidal neurons in brain slices, after photostimulation by multiple spots glutamate uncaging. By bringing extended portions of tilted dendrites simultaneously in-focus, we monitor the spatial extent of dendritic calcium signals, showing a shift from a widespread to a spatially confined response upon blockage of voltage-gated Na(+) channels.
访问大脑中的三维结构对于探测从突触输入整合到网络活动映射的多个层次的信号处理至关重要。在这里，我们提出了一种通过数字全息术与远程聚焦相结合的独立三维光激活和成像的光学方法。我们通过实验证明了在至少 300μm 的范围内对离焦成像的球差补偿，以及沿斜平面的无扫描成像。我们将该方法应用于在脑片内通过多个点的谷氨酸光解笼刺激后，对海马锥体神经元倾斜树突进行功能成像。通过将倾斜树突的扩展部分同时聚焦在焦平面上，我们监测树突钙信号的空间范围，发现阻断电压门控 Na(+)通道后，从广泛分布的反应转变为空间受限的反应。
