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）：此申请涉及广泛的挑战领域（06）启用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最广泛使用的方法之一。但是，通过衍射光学定律，空间分辨率限于约300 nm。改善空间分辨率的方法很复杂，并且并不总是与细胞成像兼容。例如，近场扫描光学显微镜（NSOM）需要接近NSOM探针与样品接触，并且只能用于对细胞的上表面进行成像。在此挑战赠款应用中，我们建议开发一种新型的显微镜技术，该技术可提供比衍射有限的光学器件好几倍的分辨率，可用于对细胞中的金属离子进行成像。我们的方法基于等离子体和纳米晶状体的新兴领域，基于金属纳米结构的新型光学性质。众所周知，在某些金属纳米结构的照明时，可能发生次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 nm之内。这种方法仅允许在细胞的底部接触区域上进行测量，该方法需要与金属结构接触，并且只能提供点测量而不是成像。接触型显微镜不能用于成像细胞内的细胞内离子浓度。现在，我们已经观察到金属纳米结构上方的一种独特现象，该现象有望在细胞的所有区域，不仅是接触区域提供亚波长成像分辨率。我们最近表明，可以在金属结构上方的实质距离上创建次波长大小的场，范围为1至10微米。我们认为，这种现象可用于激发细胞中的亚波长尺寸体积，并可以为新的一类光学显微镜提供基础。我们建议使用这种非凡的光学现象来开发一种新型显微镜，用于在空间分辨率接近50 nm的细胞中的金属离子成像。这种方法对成像金属离子的有用性将通过使用荧光寿命成像显微镜（FLIM）扩展到许多感兴趣的金属离子，这也将使影像受到光漂白的影响。通过现代纳米制作方法和计算方法的可用性，这项挑战赠款项目成为可能。我们将使用有限差分时间域方法来模拟金属纳米结构（MNS）上方的场分布。由于这些结构将用于细胞内离子成像，因此我们将计算扩展到表面上方10微米或更多。 MN的几何形状将变化以获得电场的亚波长体积。我们将对纳米荷尔阵列进行建模，该阵列将提供带有图案的照明和同心纳米阵列，从而提供点照明。我们将将此类结构称为等离子镜片。 FDTD模拟的结果将用于选择特定的几何形状进行纳米化。根据预期的波长范围，结构将由金，银或铝制成，这将延伸从紫外线到NIR的波长范围。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）光刻来制造大量逐渐变化的结构。对于更大的图案，EB比FIB快。我们将在非质量材料（例如铬）中准备类似的模式，以与金属结构进行比较。我们将使用请求的NSOM仪器来测量金属结构上方的电场分布。然后，NSOM结果将用于完善MN的几何形状，以获得最高的空间分辨率。单个斑点将分开1个或更多微米，以允许使用远场光学元件进行读数。然后，选定的金属纳米结构将与荧光和NSOM一起使用，以确定激发体积的尺寸。这些体积还将使用荧光相关光谱（FCS）估算。这些荧光测量将使用对包括Na+，K+，Ca2+，Mg2+和Zn2+的金属离子敏感的探针进行。溶液中已知的金属离子浓度将与从波长标准测量测量和FLIM确定的浓度进行比较。金属纳米结构将用于测量以下分辨率分辨率的细胞中的金属离子分布。我们选择了不同的细胞系进行成像，其中包括COS细胞，293个细胞，PC12细胞和线粒体。这些单元具有不同的特性，因此代表了一系列成像应用。强度比和寿命图像将通过栅格扫描电场阵列获得完整的蜂窝金属离子图像来获得。拟议的等离子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摄像头，以记录隔离点的图像。简单的栅格扫描将用于获得完整的图像。有或没有次波长空间分辨率，基于这种新型成像技术的这种显微镜不仅可以在细胞成像中使用，还可以使用高通量测定和医学诊断。该项目是为基于血浆和金属纳米结构开发一种新型显微镜。荧光显微镜广泛用于生物学和细胞成像中。不幸的是，空间分辨率仅限于约300 nm，这比大多数感兴趣的生物分子大得多。该项目的目的是将空间分辨率提高到约50 nm，从而大大增加了可用于细胞成像研究的信息。提出的显微镜的开发是一个具有挑战性的目标。该项目将通过雇用新科学家刺激排放。行业将利用新的显微镜技术来开发新产品。