title: Fast Calcium Imaging with Optical Sectioning via HiLo Microscopy.
abstract: Imaging intracellular calcium concentration via reporters that change their fluorescence properties upon binding of calcium, referred to as calcium imaging, has revolutionized our way to probe neuronal activity non-invasively. To reach neurons densely located deep in the tissue, optical sectioning at high rate of acquisition is necessary but difficult to achieve in a cost effective manner. Here we implement an accessible solution relying on HiLo microscopy to provide robust optical sectioning with a high frame rate in vivo. We show that large calcium signals can be recorded from dense neuronal populations at high acquisition rates. We quantify the optical sectioning capabilities and demonstrate the benefits of HiLo microscopy compared to wide-field microscopy for calcium imaging and 3D reconstruction. We apply HiLo microscopy to functional calcium imaging at 100 frames per second deep in biological tissues. This approach enables us to discriminate neuronal activity of motor neurons from different depths in the spinal cord of zebrafish embryos. We observe distinct time courses of calcium signals in somata and axons. We show that our method enables to remove large fluctuations of the background fluorescence. All together our setup can be implemented to provide efficient optical sectioning in vivo at low cost on a wide range of existing microscopes.
authors.affiliation: Wavefront-Engineering Microscopy Group, Neurophotonics Laboratory, CNRS UMR8250, University Paris Descartes, Sorbonne Paris Cité, Paris, France.
authors.forename: Marcel A
authors.initials: MA
authors.lastname: Lauterbach
authors.affiliation: Wavefront-Engineering Microscopy Group, Neurophotonics Laboratory, CNRS UMR8250, University Paris Descartes, Sorbonne Paris Cité, Paris, France.
authors.forename: Emiliano
authors.initials: E
authors.lastname: Ronzitti
authors.affiliation: Institut du Cerveau et de la Moelle Epinière (ICM), INSERM U1127, CNRS UMR 7225, Sorbonne Paris Cité, University Pierre et Marie Curie Paris 06 UMR S 1127, Paris, France.
authors.forename: Jenna R
authors.initials: JR
authors.lastname: Sternberg
authors.affiliation: Institut du Cerveau et de la Moelle Epinière (ICM), INSERM U1127, CNRS UMR 7225, Sorbonne Paris Cité, University Pierre et Marie Curie Paris 06 UMR S 1127, Paris, France.
authors.forename: Claire
authors.initials: C
authors.lastname: Wyart
authors.affiliation: Wavefront-Engineering Microscopy Group, Neurophotonics Laboratory, CNRS UMR8250, University Paris Descartes, Sorbonne Paris Cité, Paris, France.
authors.forename: Valentina
authors.initials: V
authors.lastname: Emiliani
mesh_terms: D000818:Animals; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D004625:Embryo, Nonmammalian; D008856:Microscopy, Fluorescence / Q000379:methods*; D057054:Molecular Imaging / Q000379:methods*; D009046:Motor Neurons / Q000378:metabolism*; D013116:Spinal Cord / Q000196:embryology / Q000378:metabolism; D015027:Zebrafish / Q000196:embryology / Q000378:metabolism
chemical_list: D002118:Calcium