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.

Multifocus structured illumination microscopy for fast volumetric super-resolution imaging

“…Design and manufacturing of multifocus diffractive optics has been described in detail in [10]. For a detailed description of the layout and alignment of a multifocus optical arm, see the Supplementary Material in our previous publication [11].…”

The Nanolithography Toolbox

“…Several research projects have successfully used the Toolbox, with applications ranging from the physical to the life sciences [15][16][17][18][19][20][21][22][23][24]. …”

Through-focus or volumetric type of optical imaging methods: a review

“…Much interest is given to two-photon (or multi-photon) microscopy that makes use of TF imaging to acquire 3D volumetric data of biological samples, including brain tissue and bone calcium (11, 14, 15). High speed TF imaging is used to track single-molecules in three dimensions and observe their behavior during cell division (38), and it has also been used to image entire embryos (39). By simultaneously imaging different focal planes within the sample it was possible to track the 3D dynamics in live cells at high temporal and spatial resolution (12, 13).…”