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.
Highlights
One of the main challenges in modern biological research is three-dimensional (3D) imaging of living specimens
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 lightefficiency of the central optical component of MFM: the diffractive multifocus grating (MFG)
Summary
One of the main challenges in modern biological research is three-dimensional (3D) imaging of living specimens. Light from the specimen is multiplexed by a specially designed diffractive Fourier optic – the multifocus grating (MFG) – and each beam is focus shifted so that an instant focal series is obtained, laid out in an array of 2D images of different focal planes on the camera. We here further improve the sensitivity of MFM by fabricating MFGs with eight phase levels, using a grating function design previously described in theory in a patent application by Abrahamsson and Gustafsson [12] These devices, theoretically capable of obtaining photon count efficiencies up to ≈89%, allow even faster and gentler imaging of small, sensitive or dim fluorescently labeled specimens. We report the design and implementation of a simple binary MFG design with high photon count efficiency (≈79%) for simultaneously imaging seven focal planes. We describe a “precise color” MFM layout for optimized multi-color imaging of multiple fluorophores simultaneously
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