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Proc Natl Acad Sci U S A. 2011 Dec 6;108(49):19504-9. doi: 10.1073/pnas.1109111108. Epub 2011 Nov 9.

Three-dimensional imaging and photostimulation by remote-focusing and holographic light patterning.

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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.


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.

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