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



Sara Abrahamsson, Robert Ilic, Jan Wisniewski, Brian Mehl, Liya Yu, Lei Chen, Marcelo I. Davanco, Laura Oudjedi, Jean Bernard Fiche, Bassam Hajj, Xin Jin, Joan Pulupa, Christine Cho, Mustafa Mir, Mohamed El Beheiry, Xavier Darzacq, Marcelo Nollmann, Maxime Dahan, Carl Wu, Timothee Lionnett, James Alexander Liddle, Cornelia Bargmann


Multifocus microscopy (MFM) allows high-resolution instantaneous three-dimensional (3D) imaging and has been applied to study biological specimens ranging from single molecules inside cell nuclei to entire embryos. In any live microscopy application, photon budget is a critical parameter and light-efficiency must be optimized to obtain the fastest possible frame rate while minimizing photo-damage. To address previous limitations in the light-efficiency of the central optical component of MFM - the diffractive multi-focus grating (MFG) - we here describe the design and nanofabrication process of MFGs with theoretical efficiencies up to 90 %, obtaining efficiencies of > 80 % in devices of our own manufacture. We also implement a "perfect color" MFM layout with MFGs tailored to individual fluorophores in separate optical arms. The reported optical advancements enable faster and brighter volumetric time-lapse imaging of biological samples. We demonstrate the performance of a multi-phase MFG in 3D functional neuronal imaging in living C. elegans.
Biomedical Optics Express


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


Abrahamsson, S. , Ilic, R. , Wisniewski, J. , Mehl, B. , Yu, L. , Chen, L. , Davanco, M. , Oudjedi, L. , Fiche, J. , Hajj, B. , Jin, X. , Pulupa, J. , Cho, C. , Mir, M. , El Beheiry, M. , Darzacq, X. , Nollmann, M. , Dahan, M. , Wu, C. , Lionnett, T. , Liddle, J. and Bargmann, C. (2016), Multifocus microscopy with precise color multi-phase diffractive optics applied in functional neuronal imaging, Biomedical Optics Express, [online],, (Accessed July 21, 2024)


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Created February 29, 2016, Updated October 12, 2021