Improvement of lateral resolution and extension of depth of field in two-photon microscopy by a higher-order radially polarized beam

Sari Ipponjima, Terumasa Hibi, Yuichi Kozawa, Hibiki Horanai, Hiroyuki Yokoyama, Shunichi Sato, Tomomi Nemoto

研究成果: Article査読

24 被引用数 (Scopus)

抄録

The spatial resolution of laser scanning microscopes depends on the focal spot size. As previously reported, we successfully improved the lateral spatial resolution in confocal microscopy using liquid crystal devices (LCDs) to convert a linearly polarized (LP) beam into a higher-order radially polarized (HRP) beam. Taking advantage of the fact that those LCDs can be utilized at various wavelengths, including near-infrared, we employed a near-infrared HRP beam to improve the resolution in two-photon microscopy. Point-spread functions estimated from fluorescent beads embedded in agarose gel showed that an HRP beam at 800-nm excitation improved lateral resolution to 230 nm from 294 nm, which was obtained using an LP beam at the same wavelength. Furthermore, at the glass–water interface, the lateral resolution was considerably improved to 188 nm using the HRP beam, whereas it degraded to 510 nm while using the LP beam. The HRP beams visualized fine intracellular structures not only in fixed cells stained with various dyes but also in living cells. Moreover, the HRP beam significantly extended the depth of field, which facilitated obtaining infocus images, especially during time-lapse observations of living cells. These results indicate that our method is applicable to various biological applications.

本文言語English
ページ(範囲)23-32
ページ数10
ジャーナルMicroscopy
63
1
DOI
出版ステータスPublished - 2014 2月

ASJC Scopus subject areas

  • 構造生物学
  • 器械工学
  • 放射線学、核医学およびイメージング

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