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

Research output: Contribution to journalArticlepeer-review

23 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)23-32
Number of pages10
JournalMicroscopy
Volume63
Issue number1
DOIs
Publication statusPublished - 2014 Feb

Keywords

  • Depth of field
  • Liquid crystals
  • Multi-photon microscopy
  • Phase modulation
  • Polarization
  • Spatial resolution

ASJC Scopus subject areas

  • Structural Biology
  • Instrumentation
  • Radiology Nuclear Medicine and imaging

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