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

工程摘要 该提案旨在开发一种能够通过显微镜以80 nm横向分辨率实时成像的显微镜。 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低至10 nm，但需要10，000至100，000 原始图像，并且因此不适于快速实时成像。受激发射损耗显微镜可以 在活细胞中实现约50 nm的分辨率，并在小视场（约1 µm2）内快速成像，但由于它是一种 点扫描技术在视场增大时变得太慢。STED还要求高 强度降低了其对实时成像的吸引力。RESOLFT显微镜和相关技术 显示出有希望的结果，在活体样品中达到~ 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）是一种有吸引力的方法，用于活体超 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。线性SIM可以实现~ 120 nm的分辨率，实时成像已被 在二维和三维两个维度上进行演示，2D SIM的最快帧速率超过50 每秒帧数。非线性SIM理论上可以实现无限的分辨率和成像与40纳米 分辨率已在两个维度中得到证明。实时二维NSIM已在 2.8每秒62帧，分辨率为62 nm。 三维非线性SIM尚未得到证实。实时3D NSIM与2D的开发 超过每秒4帧的帧速率将满足在大视场上的高分辨率实时成像的需要， 视野可达~ 100 µm × 100 µm，允许细胞结构，如丝状伪足，囊泡和线粒体， 在三维空间中解决。为了实现这一目标，我们将开发一种新的结构照明模式 发生器光学设计和新颖的自适应光学技术可实现最佳光学性能 允许在细胞和薄组织切片中快速无像差成像。

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.