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* Corresponding author 1 The Rockefeller University 2 NIST - National Institute of Standards and Technology Gaithersburg 3 Janelia Farm Research Campus 4 CBS - Centre de Biochimie Structurale Montpellier 5 PCC - Physico-Chimie-Curie 6 UC Berkeley

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 : Diffractive optics Multiple imaging Polarization Three-dimensional microscopy Fluorescence microscopy





Author: Sara Abrahamsson - Rob Ilic - Jan Wisniewski - Brian Mehl - Liya Yu - Lei Chen - Marcelo Davanco - Laura Oudjedi - Jean-Bernard F

Source: https://hal.archives-ouvertes.fr/



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