Improvement of lateral resolution and extension of depth of field in two-photon microscopy by a higher-order radially polarized beam.
Improvement of lateral resolution and extension of depth of field in two-photon microscopy by a higher-order radially polarized beam.
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DOI：
10.1093/jmicro/dft041
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发表时间：
2014-02
期刊：
影响因子：
1.8
通讯作者：
Sari Ipponjima;Terumasa Hibi;Y. Kozawa;Hibiki Horanai;H. Yokoyama;S. Sato;T. Nemoto
中科院分区：
文献类型：
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作者：
Sari Ipponjima;Terumasa Hibi;Y. Kozawa;Hibiki Horanai;H. Yokoyama;S. Sato;T. Nemoto
激光扫描显微镜的空间分辨率取决于焦点尺寸。正如之前报道的，我们使用液晶器件（LCD）将线偏振（LP）光束转换为高阶径向偏振（HRP）光束，成功提高了共焦显微镜的横向空间分辨率。利用这些 LCD 可以在包括近红外在内的各种波长下使用的事实，我们采用近红外 HRP 光束来提高双光子显微镜的分辨率。根据嵌入琼脂糖凝胶中的荧光珠估计的点扩散函数表明，800 nm 激发的 HRP 光束将横向分辨率从 294 nm 提高到 230 nm，这是使用相同波长的 LP 光束获得的。此外，在玻璃-水界面，使用 HRP 光束时横向分辨率显着提高至 188 nm，而使用 LP 光束时横向分辨率下降至 510 nm。 HRP 光束不仅可以显示用各种染料染色的固定细胞中的精细细胞内结构，还可以显示活细胞中的精细细胞内结构。此外，HRP 光束显着扩展了景深，这有助于获得清晰的图像，特别是在活细胞的延时观察过程中。这些结果表明我们的方法适用于各种生物应用。
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.