Super-high resolution optical nanoscopy based on microlenses

描述(申请人提供)：从基础研究和应用观点来看，光学显微镜在生物、化学和材料科学中是一种无价的表征工具。光学显微镜的空间分辨率有一个物理限制(通过阿贝定律)，低于这个限制，光学特征就无法分辨。在这里，我们展示了由高折射率(n，1.47；n&lt；1.73)材料组成的盐基平凸微透镜(MLS)的使用，极大地提高了使用传统倒置显微镜获取的图像的空间分辨率。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光源提供x2和x6之间的放大。高分辨率图像可以在生物样本活跃的大气条件下获取。该方法成本低廉，使用方便，不需要大量的样品前处理。MLS的制造非常简单，并且可以在潮湿的实验室中自组装出高度可重复性的MLS阵列。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，包括明场和暗场、相位对比度和荧光-在大气条件下对生物样本进行成像。具体工作目标：目标1：我们计划从实验和理论上研究影响MLS成像质量的物理参数，包括放大倍率、空间分辨率和对比度。一般来说，微透镜的大小、形状、折射率和ML样品的距离都会影响微透镜的物理参数(焦距、焦斑大小、空间分辨率、放大率和焦深)。这些研究对于加深我们对光与MLS相互作用的理解，以及优化MLS在生命、生物医学和材料科学中的成像应用的性能是至关重要的。通过这一特定的目标，我们打算从实验和理论上研究ML维度、ML曲率(大小)、折射率和ML样本距离对放大倍率和空间分辨率的影响。实验结果将与基于几何光学和电磁理论的光线追踪和时域有限差分(FDTD)方法的计算结果相印证。目的2：使用优化的微透镜纳米显微镜进行成像。我们计划利用优化后的MLS在传统光学显微镜上获得超高分辨率。在目标1中获得的关于为超高分辨率优化最小二乘法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管。我们的纳米显微镜将由三个主要组件组成：传统光学显微镜、(或阵列)微透镜和压电扫描工作台。对于概念验证实验，我们将对70-150 nm尺寸范围内的蛋白质和DNA纳米阵列、噬菌体和病毒颗粒进行荧光和明场成像和扫描。最终，建议的成像工具将对科学家、工程师和临床医生有用，用于化学和生化成像、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.