A compact holographic projector module for high-resolution 3D multi-site two-photon photostimulation

dc.contributor.authorGo, Mary Ann
dc.contributor.authorMueller, Max
dc.contributor.authorCastañares, Michael Lawrence
dc.contributor.authorEgger, Veronica
dc.contributor.authorDaria, Vincent
dc.date.accessioned2019-02-20T01:25:36Z
dc.date.available2019-02-20T01:25:36Z
dc.date.issued2019-01-28
dc.date.updated2019-02-03T09:05:17Z
dc.description.abstractPatterned two-photon (2P) photolysis via holographic illumination is a powerful method to investigate neuronal function because of its capability to emulate multiple synaptic inputs in three dimensions (3D) simultaneously. However, like any optical system, holographic projectors have a finite space-bandwidth product that restricts the spatial range of patterned illumination or field-of-view (FOV) for a desired resolution. Such trade-off between holographic FOV and resolution restricts the coverage within a limited domain of the neuron’s dendritic tree to perform highly resolved patterned 2P photolysis on individual spines. Here, we integrate a holographic projector into a commercial 2P galvanometer-based 2D scanning microscope with an uncaging unit and extend the accessible holographic FOV by using the galvanometer scanning mirrors to reposition the holographic FOV arbitrarily across the imaging FOV. The projector system utilizes the microscope’s built-in imaging functions. Stimulation positions can be selected from within an acquired 3D image stack (the volume-of-interest, VOI) and the holographic projector then generates 3D illumination patterns with multiple uncaging foci. The imaging FOV of our system is 800×800 μm2 within which a holographic VOI of 70×70×70 μm3 can be chosen at arbitrary positions and also moved during experiments without moving the sample. We describe the design and alignment protocol as well as the custom software plugin that controls the 3D positioning of stimulation sites. We demonstrate the neurobiological application of the system by simultaneously uncaging glutamate at multiple spines within dendritic domains and consequently observing summation of postsynaptic potentials at the soma, eventually resulting in action potentials. At the same time, it is possible to perform two-photon Ca2+ imaging in 2D in the dendrite and thus to monitor synaptic Ca2+ entry in selected spines and also local regenerative events such as dendritic action potentials.en_AU
dc.description.sponsorshipE received funding from the German Federal Ministry of Education and Research (BMBF) contract number FKZ 01GQ1502; VD received funding from the Australian Research Council DP140101555.en_AU
dc.format.mimetypeapplication/pdfen_AU
dc.identifier.issn1932-6203en_AU
dc.identifier.urihttp://hdl.handle.net/1885/156441
dc.language.isoen_AUen_AU
dc.provenanceCreative Commons Attribution Licenseen_AU
dc.publisherPublic Library of Scienceen_AU
dc.relationhttp://purl.org/au-research/grants/arc/DP140101555en_AU
dc.rights� 2019 Go et al.en_AU
dc.rights.licenseThis is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.en_AU
dc.rights.urihttps://creativecommons.org/licenses/by/4.0/en_AU
dc.sourcePLOS ONEen_AU
dc.titleA compact holographic projector module for high-resolution 3D multi-site two-photon photostimulationen_AU
dc.typeJournal articleen_AU
dcterms.accessRightsOpen Accessen_AU
local.bibliographicCitation.issue1en_AU
local.bibliographicCitation.lastpagee0210564-17en_AU
local.bibliographicCitation.startpagee0210564-1en_AU
local.contributor.affiliationDaria, V., Eccles Institute of Neuroscience, John Curtin School of Medical Research, The Australian National Universityen_AU
local.contributor.authoremailvincent.daria@anu.edu.auen_AU
local.contributor.authoruidu4492652en_AU
local.description.notesImported from PLOSen_AU
local.identifier.ariespublicationu3102795xPUB678
local.identifier.citationvolume14en_AU
local.identifier.doi10.1371/journal.pone.0210564en_AU
local.identifier.uidSubmittedByu1005913en_AU
local.publisher.urlhttps://www.plos.org/en_AU
local.type.statusPublished Versionen_AU

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