Format

Send to

Choose Destination
Biomed Opt Express. 2016 Feb 16;7(3):855-69. doi: 10.1364/BOE.7.000855. eCollection 2016 Mar 1.

Multifocus microscopy with precise color multi-phase diffractive optics applied in functional neuronal imaging.

Author information

1
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.
2
Center for Nanoscale Science and Technology, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA.
3
Transcription Imaging Consortium, HHMI Janelia Research Campus, Ashburn, VA 20147, USA.
4
Centre de Biochimie Structurale, CNRS UMR5048, INSERM U1054, Université de Montpellier, 29 rue de Navacelles, 34090 Montpellier, France.
5
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.
6
HHMI and Lulu and Anthony Wang Laboratory of Neural Circuits and Behavior, The Rockefeller University, New York, NY 10065, USA.
7
Laboratory of Cellular Biophysics, The Rockefeller University, New York, NY 10065, USA.
8
Transcription Imaging Consortium, HHMI Janelia Research Campus, Ashburn, VA 20147, USA; University of California, Berkeley, CA 94720, USA.

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.

KEYWORDS:

(050.1970) Diffractive optics; (110.4190) Multiple imaging; (180.2520) Fluorescence microscopy; (180.6900) Three-dimensional microscopy; (260.5430) Polarization

Supplemental Content

Full text links

Icon for PubMed Central
Loading ...
Support Center