Nyquist-exceeding high voxel rate acquisition in mesoscopic multiphoton microscopy for full-field submicron resolution resolvability

Bhaskar Jyoti Borah; Jye-Chang Lee; Han-Hsiung Chi; Yang-Ting Hsiao; Chen-Tung Yen; Chi-Kuang Sun

doi:10.1016/j.isci.2021.103041

Nyquist-exceeding high voxel rate acquisition in mesoscopic multiphoton microscopy for full-field submicron resolution resolvability

iScience. 2021 Aug 27;24(9):103041. doi: 10.1016/j.isci.2021.103041. eCollection 2021 Sep 24.

Authors

Bhaskar Jyoti Borah¹, Jye-Chang Lee², Han-Hsiung Chi², Yang-Ting Hsiao¹, Chen-Tung Yen², Chi-Kuang Sun^{1

3

4}

Affiliations

¹ Department of Electrical Engineering and Graduate Institute of Photonics and Optoelectronics, National Taiwan University, Taipei 10617, Taiwan.
² Department of Life Science, National Taiwan University, Taipei 10617, Taiwan.
³ Graduate Institute of Biomedical Electronics and Bioinformatics, National Taiwan University, Taipei 10617, Taiwan.
⁴ Molecular Imaging Center, National Taiwan University, Taipei 10617, Taiwan.

Abstract

The Nyquist-Shannon criterion has never been realized in a laser-scanning mesoscopic multiphoton microscope (MPM) with a large field-of-view (FOV)-resolution ratio, especially when employing a high-frequency resonant-raster-scanning. With a high optical resolution nature, a current mesoscopic-MPM either neglects the criterion and degrades the digital resolution to twice the pixel size, or reduces the FOV and/or the raster-scanning speed to avoid aliasing. We introduce a Nyquist figure-of-merit (NFOM) parameter to characterize a laser-scanning MPM in terms of its optical-resolution retrieving ability. Based on NFOM, we define the maximum aliasing-free FOV, and subsequently, a cross-over excitation wavelength, below which the FOV becomes NFOM-constrained irrespective of an optimized optical design. We validate our idea in a custom-built mesoscopic-MPM with millimeter-scale FOV yielding an ultra-high FOV-resolution ratio of >3,000, while securing up-to a 1.6 mm Nyquist-satisfied aliasing-free FOV, a ∼400 nm lateral resolution, and a 70 M/s effective voxel-sampling rate, all at the same time.

Keywords: Data acquisition in bioinformatics; Materials science; Optical imaging.