title: Improvement of lateral resolution and extension of depth of field in two-photon microscopy by a higher-order radially polarized beam.
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 in-focus images, especially during time-lapse observations of living cells. These results indicate that our method is applicable to various biological applications.
authors.affiliation: Research Institute for Electronic Science, Hokkaido University, Kita 21 Nishi 10, Kita-ku, Sapporo 001-0021, Japan.
authors.forename: Sari
authors.initials: S
authors.lastname: Ipponjima
authors.forename: Terumasa
authors.initials: T
authors.lastname: Hibi
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Kozawa
authors.forename: Hibiki
authors.initials: H
authors.lastname: Horanai
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Yokoyama
authors.forename: Shunichi
authors.initials: S
authors.lastname: Sato
authors.forename: Tomomi
authors.initials: T
authors.lastname: Nemoto
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D018613:Microscopy, Confocal / Q000379:methods*; D017785:Photons
keywords: depth of field; liquid crystals; multi-photon microscopy; phase modulation; polarization; spatial resolution