title: Computer-generated holography enhances voltage dye fluorescence discrimination in adjacent neuronal structures.
abstract: Voltage-sensitive fluorescence indicators enable tracking neuronal electrical signals simultaneously in multiple neurons or neuronal subcompartments difficult to access with patch electrodes. However, efficient widefield epifluorescence detection of rapid voltage fluorescence transients necessitates that imaged cells and structures lie sufficiently far from other labeled structures to avoid contamination from out of focal plane and scattered light. We overcame this limitation by exciting dye fluorescence with one-photon computer-generated holography shapes contoured to axons or dendrites of interest, enabling widefield detection of voltage fluorescence with high spatial specificity. By shaping light onto neighboring axons and dendrites, we observed that dendritic back-propagating action potentials were broader and slowly rising compared with axonal action potentials, differences not measured in the same structures illuminated with a large "pseudowidefield" (pWF) spot of the same excitation density. Shaped illumination trials showed reduced baseline fluorescence, higher baseline noise, and fractional fluorescence transient amplitudes two times greater than trials acquired with pWF illumination of the same regions.
authors.affiliation: Paris Descartes University , Biomedical and Fundamental Science Faculty, Wavefront-Engineering Microscopy Group, Neurophotonics Laboratory, CNRS UMR8250, 45, rue des Saints Pères, 75270 Paris Cedex 06, France.
authors.forename: Amanda J
authors.initials: AJ
authors.lastname: Foust
authors.affiliation: Paris Descartes University , Biomedical and Fundamental Science Faculty, Wavefront-Engineering Microscopy Group, Neurophotonics Laboratory, CNRS UMR8250, 45, rue des Saints Pères, 75270 Paris Cedex 06, France.
authors.forename: Valeria
authors.initials: V
authors.lastname: Zampini
authors.affiliation: Paris Descartes University , Biomedical and Fundamental Science Faculty, Wavefront-Engineering Microscopy Group, Neurophotonics Laboratory, CNRS UMR8250, 45, rue des Saints Pères, 75270 Paris Cedex 06, France.
authors.forename: Dimitrii
authors.initials: D
authors.lastname: Tanese
authors.affiliation: Paris Descartes University , Biomedical and Fundamental Science Faculty, Wavefront-Engineering Microscopy Group, Neurophotonics Laboratory, CNRS UMR8250, 45, rue des Saints Pères, 75270 Paris Cedex 06, France ; INSERM , 45, rue des Saints Pères, 75270 Paris Cedex 06, France.
authors.forename: Eirini
authors.initials: E
authors.lastname: Papagiakoumou
authors.affiliation: Paris Descartes University , Biomedical and Fundamental Science Faculty, Wavefront-Engineering Microscopy Group, Neurophotonics Laboratory, CNRS UMR8250, 45, rue des Saints Pères, 75270 Paris Cedex 06, France.
authors.forename: Valentina
authors.initials: V
authors.lastname: Emiliani
keywords: action potential; axon; computer-generated holography; dendrite; voltage imaging