Two-photon instant structured illumination microscopy improves the depth penetration of super-resolution imaging in thick scattering samples.
Two-photon instant structured illumination microscopy improves the depth penetration of super-resolution imaging in thick scattering samples.
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DOI：
10.1364/optica.1.000181
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发表时间：
2014-09-20
期刊：
影响因子：
10.4
通讯作者：
Shroff H
中科院分区：
文献类型：
--
作者：
Winter PW;York AG;Nogare DD;Ingaramo M;Christensen R;Chitnis A;Patterson GH;Shroff H
实现超过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具有极大的兴趣。我们描述了一种将双光子激发与结构照明显微镜（SIM）相结合的超分辨率方法，该方法能够以约1 Hz的帧率以约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，以提高分辨率，我们的显微镜允许超分辨率成像，没有额外的成本，在采集时间或光毒性相对于点扫描双光子显微镜，它是基于。我们的方法提供了更好的深度穿透和固有的光学切片比所有以前报道的超分辨率SIM实现，使超分辨率成像深度超过100 μm的盖玻片表面。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，证明了我们的系统在高分辨率下询问厚活标本的能力。
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.