Super-high resolution optical nanoscopy based on microlenses

基于微透镜的超高分辨率光学纳米显微镜

• 批准号: 8497289
• 负责人: Punit Kohli
• 金额: $ 32.8万
• 依托单位: SOUTHERN ILLINOIS UNIVERSITY CARBONDALE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8497289
• 关键词: Affect; Bacteriophages; Biochemical; Biological; Biological Process; Biosensing Techniques; Cell surface; Chemicals; Communities; DNA; Dimensions; Electromagnetics; Engineering; Fluorescence; Image; Imaging Device; Imaging Techniques; Knowledge; Laws; Length; Life; Light; Lighting; Methods; Microscope; Microscopy; Nanoarray Analytical Device; Optics; Performance; Phase; Preparation; Process; Proteins; Published Comment; Refractive Indices; Resolution; Sampling; Scanning; Science; Scientist; Shapes; Sodium Chloride; Source; Specimen; Spottings; Staging; Time; Virion; Work; base; cellular imaging; fluorescence imaging; imaging probe; nanopattern; nanoscale; novel; particle; public health relevance; research study; theories; tool

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.

描述（由申请人提供）：光学显微镜是一个宝贵的表征工具，在生物，化学和材料科学从基础研究和应用的观点。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。我们在这里证明了使用由高折射率（n，1.47<n<1.73）材料组成的盐基平凸微透镜（ML）极大地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色光照明源提供x2和x6之间的放大率。高分辨率图像可以在生物样本活跃的大气条件下获得。所提出的方法是廉价的，易于使用，并且不需要大量的样品制备。ML的制造非常简单，并且可以在湿实验室中自组装高度可再现的ML阵列。我们提出的基于ML的纳米镜可以用于许多不同的模式，包括明场和暗场，相衬和荧光-在大气条件下的生物标本的成像。目标1：我们计划从实验和理论上研究影响成像质量的ML的物理参数，包括放大率，空间分辨率和对比度。通常，ML的尺寸、形状、折射率以及ML-试样距离影响微透镜的物理参数（焦距、焦斑尺寸、空间分辨率、放大率和焦深）。这些研究对于增强我们对光与ML相互作用的理解以及优化ML在生命科学、生物医学和材料科学中成像应用的性能至关重要。通过这个特定的目标，我们打算实验和理论研究ML尺寸，ML曲率（大小），折射率，ML标本的距离放大倍率和空间分辨率的效果。实验结果将与基于几何光学和电磁理论使用射线追踪和时域有限差分（FDTD）方法的计算相证实。目标2：使用优化的基于微透镜的纳米显微镜成像。我们计划利用优化的ML使用传统的光学显微镜获得超高分辨率。在目标1中获得的关于优化ML以获得超高分辨率的知识将用于对纳米级生物颗粒和纳米光刻制造的纳米颗粒进行成像。我们的纳米显微镜将包括三个主要组成部分：一个传统的光学显微镜，（或阵列）微透镜（es）和压电扫描阶段。对于概念验证实验，我们将在70-150 nm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明场成像和扫描。最终，所提出的成像工具将有助于科学家、工程师和临床医生进行化学和生物化学成像，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。