title: Multifocus microscopy with precise color multi-phase diffractive optics applied in functional neuronal imaging.
abstract: Multifocus microscopy (MFM) allows high-resolution instantaneous three-dimensional (3D) imaging and has been applied to study biological specimens ranging from single molecules inside cells nuclei to entire embryos. We here describe pattern designs and nanofabrication methods for diffractive optics that optimize the light-efficiency of the central optical component of MFM: the diffractive multifocus grating (MFG). We also implement a "precise color" MFM layout with MFGs tailored to individual fluorophores in separate optical arms. The reported advancements enable faster and brighter volumetric time-lapse imaging of biological samples. In live microscopy applications, photon budget is a critical parameter and light-efficiency must be optimized to obtain the fastest possible frame rate while minimizing photodamage. We provide comprehensive descriptions and code for designing diffractive optical devices, and a detailed methods description for nanofabrication of devices. Theoretical efficiencies of reported designs is ≈90% and we have obtained efficiencies of > 80% in MFGs of our own manufacture. We demonstrate the performance of a multi-phase MFG in 3D functional neuronal imaging in living C. elegans.
authors.affiliation: HHMI and Lulu and Anthony Wang Laboratory of Neural Circuits and Behavior, The Rockefeller University, New York, NY 10065, USA; Center for Nanoscale Science and Technology, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA.
authors.forename: Sara
authors.initials: S
authors.lastname: Abrahamsson
authors.affiliation: Center for Nanoscale Science and Technology, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA.
authors.forename: Rob
authors.initials: R
authors.lastname: Ilic
authors.affiliation: Transcription Imaging Consortium, HHMI Janelia Research Campus, Ashburn, VA 20147, USA.
authors.forename: Jan
authors.initials: J
authors.lastname: Wisniewski
authors.affiliation: Transcription Imaging Consortium, HHMI Janelia Research Campus, Ashburn, VA 20147, USA.
authors.forename: Brian
authors.initials: B
authors.lastname: Mehl
authors.affiliation: Center for Nanoscale Science and Technology, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA.
authors.forename: Liya
authors.initials: L
authors.lastname: Yu
authors.affiliation: Center for Nanoscale Science and Technology, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA.
authors.forename: Lei
authors.initials: L
authors.lastname: Chen
authors.affiliation: Center for Nanoscale Science and Technology, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA.
authors.forename: Marcelo
authors.initials: M
authors.lastname: Davanco
authors.affiliation: Centre de Biochimie Structurale, CNRS UMR5048, INSERM U1054, Université de Montpellier, 29 rue de Navacelles, 34090 Montpellier, France.
authors.forename: Laura
authors.initials: L
authors.lastname: Oudjedi
authors.affiliation: Centre de Biochimie Structurale, CNRS UMR5048, INSERM U1054, Université de Montpellier, 29 rue de Navacelles, 34090 Montpellier, France.
authors.forename: Jean-Bernard
authors.initials: JB
authors.lastname: Fiche
authors.affiliation: Transcription Imaging Consortium, HHMI Janelia Research Campus, Ashburn, VA 20147, USA; Laboratoire Physico Chimie, Institut Curie, CNRS UMR 168, Université Pierre et Marie Curie-Paris 6, 11 rue Pierre et Marie Curie, 75005 Paris France.
authors.forename: Bassam
authors.initials: B
authors.lastname: Hajj
authors.affiliation: HHMI and Lulu and Anthony Wang Laboratory of Neural Circuits and Behavior, The Rockefeller University, New York, NY 10065, USA.
authors.forename: Xin
authors.initials: X
authors.lastname: Jin
authors.affiliation: Laboratory of Cellular Biophysics, The Rockefeller University, New York, NY 10065, USA.
authors.forename: Joan
authors.initials: J
authors.lastname: Pulupa
authors.affiliation: HHMI and Lulu and Anthony Wang Laboratory of Neural Circuits and Behavior, The Rockefeller University, New York, NY 10065, USA.
authors.forename: Christine
authors.initials: C
authors.lastname: Cho
authors.affiliation: Transcription Imaging Consortium, HHMI Janelia Research Campus, Ashburn, VA 20147, USA; University of California, Berkeley, CA 94720, USA.
authors.forename: Mustafa
authors.initials: M
authors.lastname: Mir
authors.affiliation: Transcription Imaging Consortium, HHMI Janelia Research Campus, Ashburn, VA 20147, USA; Laboratoire Physico Chimie, Institut Curie, CNRS UMR 168, Université Pierre et Marie Curie-Paris 6, 11 rue Pierre et Marie Curie, 75005 Paris France.
authors.forename: Mohamed
authors.initials: M
authors.lastname: El Beheiry
authors.affiliation: Transcription Imaging Consortium, HHMI Janelia Research Campus, Ashburn, VA 20147, USA; University of California, Berkeley, CA 94720, USA.
authors.forename: Xavier
authors.initials: X
authors.lastname: Darzacq
authors.affiliation: Centre de Biochimie Structurale, CNRS UMR5048, INSERM U1054, Université de Montpellier, 29 rue de Navacelles, 34090 Montpellier, France.
authors.forename: Marcelo
authors.initials: M
authors.lastname: Nollmann
authors.affiliation: Transcription Imaging Consortium, HHMI Janelia Research Campus, Ashburn, VA 20147, USA; Laboratoire Physico Chimie, Institut Curie, CNRS UMR 168, Université Pierre et Marie Curie-Paris 6, 11 rue Pierre et Marie Curie, 75005 Paris France.
authors.forename: Maxime
authors.initials: M
authors.lastname: Dahan
authors.affiliation: Transcription Imaging Consortium, HHMI Janelia Research Campus, Ashburn, VA 20147, USA.
authors.forename: Carl
authors.initials: C
authors.lastname: Wu
authors.affiliation: Transcription Imaging Consortium, HHMI Janelia Research Campus, Ashburn, VA 20147, USA.
authors.forename: Timothée
authors.initials: T
authors.lastname: Lionnet
authors.affiliation: Center for Nanoscale Science and Technology, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA.
authors.forename: J Alexander
authors.initials: JA
authors.lastname: Liddle
authors.affiliation: HHMI and Lulu and Anthony Wang Laboratory of Neural Circuits and Behavior, The Rockefeller University, New York, NY 10065, USA.
authors.forename: Cornelia I
authors.initials: CI
authors.lastname: Bargmann
keywords: (050.1970) Diffractive optics; (110.4190) Multiple imaging; (180.2520) Fluorescence microscopy; (180.6900) Three-dimensional microscopy; (260.5430) Polarization