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扩展焦点深度 两光子光板显微镜用于在细胞分辨率下进行大型多细胞生物的体内光成像
Takashi Saitou1,2, Takeshi Imamura1,2
1Department of Molecular Medicine for Pathogenesis, Graduate School of Medicine, Ehime University, Toon 791-0295, Ehime, Japan.
International journal of molecular sciences
|June 28, 2023
概括
这项研究增强了双光子光片显微镜,用于对大型生物体的实时成像. 一种新的焦点深度扩展方法提高了亚细胞分辨率,同时保持了广的视野.
科学领域:
- 生物医学工程 生物医学工程
- 显微镜的使用方法
- 光学成像技术的成像
背景情况:
- 双光子贝塞尔束光片显微镜使多细胞生物体的实时成像成为可能.
- 之前的系统使用10倍放大和NA 0.5.5实现了1毫米视野和4μm轴分辨率.
- 对于先进的实时成像应用,需要更高的分辨率.
研究的目的:
- 开发一种具有增强分辨率和大视野的光板显微镜.
- 调查用于改善照明和检测的焦点深度扩展方法.
- 在大型标本的实时成像中实现亚细胞分辨率.
主要方法:
- 使用16倍放大和高NA 0.8镜头构建了一个光片显微镜.
- 实现了一个有五个环形区域的梯级设备,以扩展焦点深度 (DOF).
- 使用光珠评估分辨率,并将系统应用于medaka鱼的体内成像.
主要成果:
- 扩展的DOF系统成功地覆盖了光板厚度,使DOF翻了一番.
- 解析度测量显示,尽管扩展了DOF,但最小的减少.
- 麦达卡鱼的体内成像表明,它可以弥补远部位的图像质量下降.
结论:
- 扩展的DOF系统有效地与宽场两光子光片显微镜相结合.
- 这种设置为大型多细胞生物体的实时成像提供了一种简单且易于使用的方法.
- 复杂的生物样本可以达到亚细胞分辨率.
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