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Two-photon instant structured illumination microscopy improves the depth penetration of super-resolution imaging in thick scattering samples.
Optica ( IF 10.4 ) Pub Date : 2014-12-09 , DOI: 10.1364/optica.1.000181
Peter W Winter 1 , Andrew G York 1 , Damian Dalle Nogare 2 , Maria Ingaramo 3 , Ryan Christensen 1 , Ajay Chitnis 2 , George H Patterson 3 , Hari Shroff 1
Affiliation  

Fluorescence imaging methods that achieve spatial resolution beyond the diffraction limit (super-resolution) are of great interest in biology. We describe a super-resolution method that combines two-photon excitation with structured illumination microscopy (SIM), enabling three-dimensional interrogation of live organisms with ~150 nm lateral and ~400 nm axial resolution, at frame rates of ~1 Hz. By performing optical rather than digital processing operations to improve resolution, our microscope permits super-resolution imaging with no additional cost in acquisition time or phototoxicity relative to the point-scanning two-photon microscope upon which it is based. Our method provides better depth penetration and inherent optical sectioning than all previously reported super-resolution SIM implementations, enabling super-resolution imaging at depths exceeding 100 μm from the coverslip surface. The capability of our system for interrogating thick live specimens at high resolution is demonstrated by imaging whole nematode embryos and larvae, and tissues and organs inside zebrafish embryos.

中文翻译:

双光子即时结构化照明显微镜可提高厚散射样品中超分辨率成像的深度穿透能力。

实现超出衍射极限的空间分辨率(超分辨率)的荧光成像方法在生物学中引起了极大的兴趣。我们描述了一种超分辨率方法,该方法将双光子激发与结构化照明显微镜(SIM)相结合,可在约1 Hz的帧频下以约150 nm的横向分辨率和约400 nm的轴向分辨率对活生物体进行三维询问。通过执行光学而不是数字处理操作来提高分辨率,相对于其所基于的点扫描两光子显微镜,我们的显微镜可以进行超分辨率成像,而无需花费额外的获取时间或光毒性。与以前报道的所有超分辨率SIM实现相比,我们的方法可提供更好的深度穿透和固有的光学切片效果,能够在距盖玻片表面超过100μm的深度进行超分辨率成像。通过对整个线虫胚胎和幼虫以及斑马鱼胚胎内的组织和器官进行成像,可以证明我们的系统能够以高分辨率询问厚的活体标本的能力。
更新日期:2019-11-01
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