Three-dimensional Nonlinear Structured Illumination for Live Imaging with 80 nm resolution

项目摘要 这项提议旨在开发一种能够通过 三维非线性结构照明显微镜的发展。单分子 定位显微镜可以提供前所未有的分辨率，分辨率可达10纳米，但需要10,000到100,000 原始图像，因此，不服从快速实时成像。受激发射耗尽显微镜可以 在活细胞中实现约50 nm的分辨率，并在小视场(约1微米)下快速成像，但因为它是 当视场增大时，点扫描技术变得太慢。STED还要求高 强度降低了它对实时成像的吸引力。RESOLFT显微镜和相关技术已经 显示了令人振奋的结果，在活样本中达到了~80 nm的分辨率，但也很慢，因为它们需要 横向样品扫描。结构照明显微镜(SIM)是一种有吸引力的现场超视场成像技术 分辨率成像因为它需要相对较少的原始图像，不需要样品的横向扫描， 并且激发强度可以很低。线阵SIM可以达到~120 nm的分辨率，实时成像已经 在二维和三维两个维度上演示，对于50以上的2D SIM卡具有最快的帧速率 每秒的帧数。理论上，非线性SIM可以实现40 nm的无限分辨率和成像 分辨率已经在两个维度上得到了证明。现场二维NSIM已在 每秒2.8帧，分辨率为62 nm。 三维非线性SIM尚未得到验证。带2D的实时3D NSIM的开发 超过每秒4帧的帧速率将满足在大视场上进行高分辨率实时成像的需求 最大可查看~100微米×100微米，允许细胞结构，如丝状孢子、小泡和线粒体 在三维空间中进行解析。为了实现这一点，我们将开发一种新的结构化照明模式 发电机光学设计和新颖的自适应光学技术，实现最佳的光学性能 允许在细胞和薄组织切片中进行快速无像差成像。

Project Abstract This proposal seeks to develop a microscope capable of live imaging with 80 nm lateral resolution through the development of Three-dimensional Nonlinear Structured Illumination Microscopy. Single Molecule Localization Microscopy can offer unprecedented resolution down to 10 nm but requires 10,000 to 100,000 raw images and, therefore, is not amenable to fast live imaging. Stimulated Emission Depletion Microscopy can achieve ~ 50 nm resolution in live cells and rapid imaging over small (~1 µm2) fields of view but because it is a point scanning technique becomes too slow when the field of view increases. STED also requires high intensities reducing its attractiveness for live imaging. RESOLFT microscopy and associated techniques have shown promising results, achieving ~ 80 nm resolution in live samples but are also slow because they require lateral sample scanning. Structured Illumination Microscopy (SIM) is an attractive approach for live super- resolution imaging because it requires relatively few raw images, no lateral scanning of the sample is required, and the excitation intensity can be low. Linear SIM can achieve ~ 120 nm resolution and live imaging has been demonstrated in both two dimensions and three dimensions with the fastest frame rates for 2D SIM over 50 frames per second. Nonlinear SIM can theoretically achieve unlimited resolution and imaging with 40 nm resolution has been demonstrated in two dimensions. Live two-dimensional NSIM has been demonstrated at 2.8 frames per second with 62 nm resolution. Three-dimensional Nonlinear SIM has yet to be demonstrated. The development of live 3D NSIM with 2D frame rates exceeding 4 frames per second will fill a need for high-resolution live imaging over large fields of view up to ~ 100 µm × 100 µm, allowing cellular structures such as filopodia, vesicles, and mitochondria to be resolved in three-dimensions. To accomplish this we will develop a new Structured Illumination Pattern generator optical design and novel Adaptive Optics Techniques to achieve the best optical performance allowing fast aberration-free imaging in cells and thin tissue sections.