Sub-Wavelength Imaging of Intracellular Metal Ions

细胞内金属离子的亚波长成像

• 批准号: 7940807
• 负责人: Joseph R. LAKOWICZ
• 金额: $ 36.12万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7940807
• 关键词: Address; Affect; Aluminum; Applications Grants; Area; Binding; Biological Sciences; Biology; COS Cells; Cell Line; Cell surface; Cells; Chromium; Complex; Computing Methodologies; Development; Diagnostic; Dimensions; Electron Beam; Fluorescence; Fluorescence Microscopy; Fluorescence Spectroscopy; Goals; Gold; Grant; Growth; Image; Imaging Techniques; Individual; Industry; Ions; Laws; Life; Light; Lighting; Measurement; Measures; Medical; Metals; Methods; Microscope; Microscopy; Mitochondria; Modeling; Nanostructures; Neurons; Optics; PC12 Cells; Pattern; Photobleaching; Property; Research; Resolution; Sampling; Scanning; Science; Scientist; Silver; Simulate; Solutions; Specificity; Spectrum Analysis; Spottings; Structure; Surface; System; Techniques; Technology; Time; aqueous; base; biological research; cellular imaging; charge coupled device camera; design; design and construction; electric field; electron beam lithography; fluorescence imaging; fluorophore; high throughput screening; improved; instrument; interest; lens; nano; nanofabrication; near field microscopy; novel; particle; plasmonics; ratiometric; simulation; theories

DESCRIPTION (provided by applicant): This application addresses broad the Challenge Area (06) Enabling Technologies and Specific Challenge Topic 06-GM-106; subcellular imaging of metal ions. Fluorescence microscopy is one of the most widely used methods in the biological sciences. However, the spatial resolution is limited to about 300 nm by the laws of diffractive optics. Methods to improve the spatial resolution are complex and not always compatible with cell imaging. For example, near-field scanning optical microscopy (NSOM) requires near contact of the NSOM probe with the sample and can only be used to image the upper surface of cells. In this Challenge Grant application we propose to develop a novel type of microscopy technique that provides resolution several-fold better than diffraction limited optics and can be used to image metal ions in cells. Our approach is based on the emerging fields of plasmonic and nano-optics, on the novel optical properties of metallic nanostructures. It is known that sub- wavelength size electric field distributions can occur upon illumination of certain metallic nanostructures. Previous approaches to use these structures for imaging were based on contact of the sample with near-fields on the metal structure, typically within 50 nm. Such methods only allow measurements on the bottom contact region of the cell, which would need to be in contact with the metallic structure, and would only provide point measurements and not imaging. Contact-type microscopy cannot be used to image the intracellular ion concentration in cells. We have now observed a unique phenomenon above metallic nanostructures which promises to provide sub-wavelength imaging resolution in all regions of the cell, not just the contact region. We have recently shown that sub-wavelength size fields can be created at substantial distances above the metallic structures, ranging from 1 to 10 microns. We believe this phenomenon can be used to excite sub-wavelength size volumes in cells and can provide the basis for a new class of optical microscopes. We propose to use this remarkable optical phenomenon to develop a novel microscope for imaging of metal ions in cells with a spatial resolution approaching 50 nm. The usefulness of this approach to imaging metal ions will be extended to many metal ions of interest by using fluorescence lifetime imaging microscopy (FLIM), which will also make the imaging to be less affected by photo bleaching. This Challenge Grant project is made possible by the availability of modern nanofabrication methods and computational methods. We will use the finite-difference time-domain method to simulate the field distributions above metallic nanostructures (MNS). Since these structures will be used for intracellular ion imaging we will extend the calculations to 10 microns or more above the surface. The geometry of the MNS will be varied to obtain sub-wavelength volumes for the electric field. We will model nanohole arrays which will provide a patterned illumination and concentric nanorings which provide point illumination. We will refer to such structures as plasmonic lenses. The results from the FDTD simulations will be used to select specific geometries for nanofabrication. Depending upon the intended wavelength range the structures will be made out of gold, silver or aluminum, which will extend the wavelength range from the UV to the NIR. We will use the focused ion beam (FIB) method to fabricate the initial structures. As needed we will use electron beam (EB) lithography to fabricate a larger number of progressively varying structures. EB is faster than FIB for larger patterns. We will prepare similar patterns in non-plasmonic materials like chromium to compare with the metal structure. We will use the requested NSOM instrument to measure the electric field distributions above the metallic structures. The NSOM results will then be used to refine the geometries of the MNS to obtain the highest spatial resolution. The individual spots will be 1 or more microns apart to allow for readout using far-field optics. The selected metal nanostructures will then be used with fluorescence and NSOM to determine the sizes of the excited volumes. These volumes will also be estimated using fluorescence correlation spectroscopy (FCS). These fluorescence measurements will be performed using probes which are sensitive to metal ions including Na+, K+, Ca2+, Mg2+ and Zn2+. The known metal ion concentrations in the solution will be compared with the concentrations determined from the wavelength-radiometric measurements and FLIM. The metallic nanostructures will be used to measure metal ion distributions in cells with sub-wavelength resolution. We selected the different cell lines for imaging, including COS cells, 293 cells, PC12 cells and mitochondria therein. These cells have different properties and thus represent a range of imaging applications. Intensity ratio and lifetime images will be obtained by raster scanning the electric field arrays to obtain complete cellular metal ion images. The proposed plasmonic microscope will have a profound impact on research in the biosciences. These microscopes will be based on an inexpensive metallic structures and a CCD camera to record the images of the isolated spots. Simple raster scanning will be used to obtain the complete image. With or without sub-wavelength spatial resolution, such microscopes based on this novel imaging technique will find use not only in cell imaging, but also with high-throughput assays and medical diagnostics. This project is to develop a new type of microscope based on plasmonics and metallic nanostructures. Fluorescence microscopy is widely used in biology and cell imaging. Unfortunately, the spatial resolution is limited to about 300 nm, which is much larger than most biomolecules of interest. The goal of this project is to increase the spatial resolution to about 50 nm, and thereby greatly increase the information available for cell imaging research. Development of the proposed microscope is a challenging goal. This project will stimulate the emission by the hiring of new scientists. The new microscope technology will be utilized by industry to develop new products.

描述（由申请人提供）：本申请涉及广泛的挑战领域（06）使能技术和特定挑战主题06- gm -106；金属离子的亚细胞成像。荧光显微镜是生物科学中应用最广泛的方法之一。然而，空间分辨率受衍射光学定律的限制在300纳米左右。提高空间分辨率的方法是复杂的，并不总是与细胞成像兼容。例如，近场扫描光学显微镜（NSOM）需要NSOM探针与样品近距离接触，并且只能用于对细胞的上表面成像。在这个挑战拨款申请中，我们建议开发一种新型显微镜技术，提供比衍射限制光学好几倍的分辨率，可用于对细胞中的金属离子成像。我们的方法是基于等离子体和纳米光学的新兴领域，基于金属纳米结构的新型光学特性。已知在某些金属纳米结构的照射下，可产生亚波长大小的电场分布。以前使用这些结构进行成像的方法是基于样品与金属结构上的近场接触，通常在50纳米范围内。这种方法只允许在电池的底部接触区域进行测量，这需要与金属结构接触，并且只能提供点测量而不能成像。接触型显微镜不能用于细胞内离子浓度的成像。我们现在已经在金属纳米结构上观察到一种独特的现象，这种现象有望在细胞的所有区域提供亚波长成像分辨率，而不仅仅是接触区域。我们最近已经证明，亚波长大小的场可以在金属结构上方的相当距离上产生，范围从1到10微米。我们相信这种现象可以用来激发细胞中亚波长大小的体积，并可以为新型光学显微镜提供基础。我们建议利用这一显著的光学现象来开发一种新型显微镜，用于细胞中金属离子的成像，其空间分辨率接近50纳米。通过使用荧光寿命成像显微镜（FLIM），这种方法对金属离子成像的有用性将扩展到许多感兴趣的金属离子，这也将使成像受光漂白的影响较小。由于现代纳米制造方法和计算方法的可用性，这个挑战基金项目成为可能。本文将采用时域有限差分方法模拟金属纳米结构（MNS）表面的场分布。由于这些结构将用于细胞内离子成像，我们将把计算扩展到表面以上10微米或更多。将改变MNS的几何形状以获得电场的亚波长体积。我们将模拟提供图案照明的纳米孔阵列和提供点照明的同心纳米孔。我们将把这种结构称为等离子体透镜。时域有限差分模拟的结果将用于选择纳米制造的特定几何形状。根据预期的波长范围，这些结构将由金、银或铝制成，这将把波长范围从紫外延伸到近红外。我们将使用聚焦离子束（FIB）方法来制造初始结构。根据需要，我们将使用电子束（EB）光刻技术来制造更多数量的逐渐变化的结构。对于较大的模式，EB比FIB更快。我们将在铬等非等离子体材料中制备类似的图案，与金属结构进行比较。我们将使用要求的NSOM仪器测量金属结构上方的电场分布。然后，NSOM的结果将用于改进MNS的几何形状，以获得最高的空间分辨率。单个点将相距1微米或更多微米，以允许使用远场光学读出。选定的金属纳米结构将与荧光和NSOM一起使用，以确定激发体积的大小。这些体积也将使用荧光相关光谱（FCS）进行估计。这些荧光测量将使用对金属离子敏感的探针进行，包括Na+， K+, Ca2+， Mg2+和Zn2+。溶液中已知的金属离子浓度将与波长辐射测量和FLIM测定的浓度进行比较。金属纳米结构将用于测量亚波长分辨率的电池中的金属离子分布。我们选择不同的细胞系进行成像，包括COS细胞、293细胞、PC12细胞和其中的线粒体。这些细胞具有不同的特性，因此代表了一系列成像应用。通过栅格扫描电场阵列获得强度比和寿命图像，从而获得完整的细胞金属离子图像。提出的等离子体显微镜将对生物科学的研究产生深远的影响。这些显微镜将基于廉价的金属结构和CCD相机来记录孤立点的图像。简单的光栅扫描将被用来获得完整的图像。无论是否具有亚波长空间分辨率，这种基于这种新型成像技术的显微镜不仅可以用于细胞成像，还可以用于高通量分析和医学诊断。本项目旨在开发一种基于等离子体和金属纳米结构的新型显微镜。荧光显微镜在生物学和细胞成像中有着广泛的应用。不幸的是，空间分辨率被限制在300纳米左右，这比大多数感兴趣的生物分子要大得多。该项目的目标是将空间分辨率提高到50纳米左右，从而大大增加细胞成像研究的可用信息。所提出的显微镜的开发是一个具有挑战性的目标。这个项目将通过雇用新的科学家来刺激排放。新的显微镜技术将被工业用于开发新产品。