Super-high resolution optical nanoscopy based on microlenses

描述（申请人提供）：从基础研究和应用的角度来看，光学显微镜是生物，化学和材料科学中宝贵的表征工具。在光学显微镜的空间分辨率上有一个物理限制（通过阿贝定律），低于这个限制，光学特征就不能被解决。我们在这里展示了使用由高折射率（n, 1.47<n<1.73）材料组成的盐基平凸微透镜（MLs）大大提高了使用传统倒置显微镜获得的图像的空间分辨率。所提出的成像技术可以分辨100 nm以下的特征，并使用低强度宽带白光照明源提供x2到x6之间的放大倍率。在生物样品活跃的大气条件下可以获得高分辨率图像。所提出的方法价格低廉，易于使用，并且不需要大量的样品制备。MLs的制造非常简单，并且可以在湿实验室中自组装一系列高度可复制的MLs。我们提出的基于ml的纳米显微镜可以在许多不同的模式下使用，包括明暗场、相衬和荧光，用于在大气条件下对生物标本进行成像。目标1：我们计划从实验和理论上研究影响成像质量的MLs物理参数，包括放大倍率、空间分辨率和对比度。一般情况下，微透镜的尺寸、形状、折射率和ml -试样距离会影响微透镜的物理参数（焦距、焦斑大小、空间分辨率、放大倍率和聚焦深度）。这些研究对于增强我们对光与MLs相互作用的理解，以及优化MLs在生命科学、生物医学和材料科学成像应用中的性能至关重要。通过这一特定目标，我们打算从实验和理论上研究ML尺寸、ML曲率（尺寸）、折射率和ML标本距离对放大倍率和空间分辨率的影响。实验结果将通过基于几何光学和电磁理论的计算，利用光线追踪和时域有限差分（FDTD）方法进行验证。目标2：利用优化的基于微透镜的纳米显微镜成像。我们计划利用优化后的质谱仪在传统光学显微镜上获得超高分辨率。在目标1中获得的关于超高分辨率MLs优化的知识将用于成像纳米级生物颗粒和纳米光刻制造的纳米图案。我们的纳米显微镜将由三个主要部件组成：一个传统的光学显微镜，一个（或一个阵列）微透镜和一个压电扫描台。对于概念验证实验，我们将在70-150纳米尺寸范围内对蛋白质和DNA纳米阵列、噬菌体和病毒颗粒进行荧光和亮场成像和扫描。最终，该成像工具将为科学家、工程师和临床医生提供有用的化学和生化成像，探测细胞表面或附近的细胞动力学和过程，以及生物传感应用。

DESCRIPTION (provided by applicant): Optical microscopy is an invaluable characterization tool in biological, chemical, and materials sciences from both fundamental studies and applied viewpoints. There is a physical limitation imposed on the spatial resolution of an optical microscope (through Abbe's law) below which the optical features cannot be resolved. We demonstrate here the use of salt-based plano-convex microlenses (MLs) composed of high-refractive index (n, 1.47<n<1.73) materials greatly enhances the spatial resolution of images acquired using a conventional inverted microscope. The proposed imaging technique can resolve features below 100 nm and provides magnification between x2 and x6 using a low intensity broad band white light illumination source. High-resolution images can be acquired in atmospheric conditions where biological samples are active. The proposed method is inexpensive, easy to use, and does not require extensive sample preparation. The fabrication of MLs is extremely simple and an array of highly reproducible MLs can be self-assembled in a wet-lab. Our proposed ML-based nanoscope can be used in many different modes including bright- and dark-fields, phase-contrast, and fluorescence - for imaging of biological specimens under atmospheric conditions. Specific aims for the proposed work: Aim 1: We plan to experimentally and theoretically investigate the physical parameters of MLs that affect the imaging quality including magnification, spatial resolution and contrast. In general, the size, shape, refractive index of MLs, and ML-specimen distance affect the physical parameters (focal length, focal spot size, spatial resolution, magnification and depth of focus) of the microlens. These studies are crucial for enhancing our understanding of interaction of light with MLs, and for optimizing the performance of MLs for imaging applications in life-, biomedical- and materials-sciences. Through this specific aim, we intend to experimentally and theoretically investigate the effect of ML dimension, ML curvature (size), refractive index, and ML-specimen distance on magnification and spatial resolution. The experimental results will be corroborated with calculations based on geometric optics and electromagnetic theory using ray tracing and Finite Difference Time Domain (FDTD) methods. Aim 2: Imaging using optimized microlens-based nanoscopy. We plan to utilize the optimized MLs to acquire super-high resolution using a conventional optical microscope. The knowledge gained in aim 1 on optimization of MLs for super- high resolution will be utilized for imaging nanoscale biological particles and nanolithographic fabricated nanopatterns. Our nanoscope will consist of three major components: a conventional optical microscope, a (or an array) microlens(es) and a piezoelectric scanning stage. For the proof-of-concept experiments, we will perform both fluorescence and bright- field imaging and scanning of protein and DNA nanoarrays, bacteriophage and viruses particles in the 70-150 nm dimension range. Ultimately, the proposed imaging tool would be useful for scientists, engineers, and clinicians for chemical and biochemical imaging, probing cellular dynamics and processes on or near cell surfaces, and biosensing applications.