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Journal of Biomedical Optics • Open Access

Correcting spherical aberrations in a biospecimen using a transmissive liquid crystal device in two-photon excitation laser scanning microscopy
Author(s): Ayano Tanabe; Terumasa Hibi; Sari Ipponjima; Kenji Matsumoto; Masafumi Yokoyama; Makoto Kurihara; Nobuyuki Hashimoto; Tomomi Nemoto

Paper Abstract

Two-photon excitation laser scanning microscopy has enabled the visualization of deep regions in a biospecimen. However, refractive-index mismatches in the optical path cause spherical aberrations that degrade spatial resolution and the fluorescence signal, especially during observation at deeper regions. Recently, we developed transmissive liquid-crystal devices for correcting spherical aberration without changing the basic design of the optical path in a conventional laser scanning microscope. In this study, the device was inserted in front of the objective lens and supplied with the appropriate voltage according to the observation depth. First, we evaluated the device by observing fluorescent beads in single- and two-photon excitation laser scanning microscopes. Using a 25× water-immersion objective lens with a numerical aperture of 1.1 and a sample with a refractive index of 1.38, the device recovered the spatial resolution and the fluorescence signal degraded within a depth of ±0.6  mm. Finally, we implemented the device for observation of a mouse brain slice in a two-photon excitation laser scanning microscope. An optical clearing reagent with a refractive index of 1.42 rendered the fixed mouse brain transparent. The device improved the spatial resolution and the yellow fluorescent protein signal within a depth of 0–0.54 mm.

Paper Details

Date Published: 5 August 2015
PDF: 11 pages
J. Biomed. Opt. 20(10) 101204 doi: 10.1117/1.JBO.20.10.101204
Published in: Journal of Biomedical Optics Volume 20, Issue 10
Show Author Affiliations
Ayano Tanabe, Hokkaido Univ. (Japan)
Citizen Holdings Co., Ltd. (Japan)
Terumasa Hibi, Hokkaido Univ. (Japan)
Sari Ipponjima, Hokkaido Univ. (Japan)
Kenji Matsumoto, Citizen Holdings Co., Ltd. (Japan)
Masafumi Yokoyama, Citizen Holdings Co., Ltd. (Japan)
Makoto Kurihara, Citizen Holdings Co., Ltd. (Japan)
Nobuyuki Hashimoto, Citizen Holdings Co., Ltd. (Japan)
Tomomi Nemoto, Hokkaido Univ. (Japan)

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