title: Superresolving dendritic spine morphology with STED microscopy under holographic photostimulation.
abstract: Emerging all-optical methods provide unique possibilities for noninvasive studies of physiological processes at the cellular and subcellular scale. On the one hand, superresolution microscopy enables observation of living samples with nanometer resolution. On the other hand, light can be used to stimulate cells due to the advent of optogenetics and photolyzable neurotransmitters. To exploit the full potential of optical stimulation, light must be delivered to specific cells or even parts of cells such as dendritic spines. This can be achieved with computer generated holography (CGH), which shapes light to arbitrary patterns by phase-only modulation. We demonstrate here in detail how CGH can be incorporated into a stimulated emission depletion (STED) microscope for photostimulation of neurons and monitoring of nanoscale morphological changes. We implement an original optical system to allow simultaneous holographic photostimulation and superresolution STED imaging. We present how synapses can be clearly visualized in live cells using membrane stains either with lipophilic organic dyes or with fluorescent proteins. We demonstrate the capabilities of this microscope to precisely monitor morphological changes of dendritic spines after stimulation. These all-optical methods for cell stimulation and monitoring are expected to spread to various fields of biological research in neuroscience and beyond.
authors.affiliation: University Paris Descartes , Wavefront-Engineering Microscopy Group, Neurophotonics Laboratory, CNRS UMR8250, Sorbonne Paris Cité, 45, rue des Saints Pères, Paris 75006, France.
authors.forename: Marcel Andreas
authors.initials: MA
authors.lastname: Lauterbach
authors.affiliation: University Paris Descartes , Wavefront-Engineering Microscopy Group, Neurophotonics Laboratory, CNRS UMR8250, Sorbonne Paris Cité, 45, rue des Saints Pères, Paris 75006, France.
authors.forename: Marc
authors.initials: M
authors.lastname: Guillon
authors.affiliation: University Paris Descartes , Synapic Trafficking Group, Neurophotonics Laboratory, CNRS UMR8250, Sorbonne Paris Cité, 45, rue des Saints Pères, Paris 75006, France.
authors.forename: Claire
authors.initials: C
authors.lastname: Desnos
authors.affiliation: University Paris Descartes , Synapic Trafficking Group, Neurophotonics Laboratory, CNRS UMR8250, Sorbonne Paris Cité, 45, rue des Saints Pères, Paris 75006, France.
authors.forename: Dany
authors.initials: D
authors.lastname: Khamsing
authors.affiliation: University Paris Descartes , Synapic Trafficking Group, Neurophotonics Laboratory, CNRS UMR8250, Sorbonne Paris Cité, 45, rue des Saints Pères, Paris 75006, France.
authors.forename: Zahra
authors.initials: Z
authors.lastname: Jaffal
authors.affiliation: University Paris Descartes , Synapic Trafficking Group, Neurophotonics Laboratory, CNRS UMR8250, Sorbonne Paris Cité, 45, rue des Saints Pères, Paris 75006, France.
authors.forename: François
authors.initials: F
authors.lastname: Darchen
authors.affiliation: University Paris Descartes , Wavefront-Engineering Microscopy Group, Neurophotonics Laboratory, CNRS UMR8250, Sorbonne Paris Cité, 45, rue des Saints Pères, Paris 75006, France.
authors.forename: Valentina
authors.initials: V
authors.lastname: Emiliani
keywords: STED; STED microscopy; computer generated holography; holography; photostimulation; plasticity; spine morphology; uncaging