Coupled Emission Microscopy for the Biosciences

用于生物科学的耦合发射显微镜

• 批准号: 10093077
• 负责人: Joseph R. LAKOWICZ
• 金额: $ 36.28万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-02-17 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10093077
• 关键词: Affect; Area; Binding; Biological Assay; Biological Sciences; Biology; Buffers; Centenarian; Chemicals; Chemistry; Collection; Consumption; Coupled; Coupling; DNA; Deposition; Detection; Development; Devices; Diagnostic tests; Dimensions; Electronics; Environment; Film; Flow Cytometry; Fluorescence; Geometry; Glass; Image; Immunoassay; Infrastructure; Isotopes; Light; Lighting; Location; Measurable; Measurement; Measures; Medical; Medicine; Memory; Metals; Methods; Microscope; Microscopy; Modernization; Nanoporous; Optics; Plasma Enhancement; Positioning Attribute; Preparation; Proteins; Research; Resolution; Resources; Sampling; Shapes; Slide; Source; Spottings; Structure; Surface; Testing; Thick; Thinness; aqueous; biological research; cost; cytokine; density; design; detector; experimental study; fluorescence imaging; fluorophore; imaging detector; improved; instrument; instrumentation; lens; loss of function; metallicity; microscopic imaging; multiplex assay; nanofabrication; nanoscale; portability; prevent; silicon nitride; simulation; vapor

Abstract Fluorescence imaging is used widely in almost all the biosciences. Examples include imaging of microscope slides with spots of DNA or proteins, multiplexed assays of classes of molecules like cytokines or capture immunoassays. Fluorescence occurs in all directions and it is necessary to collect as much of the emission as possible and to focus the emission onto a detector. The instruments for these measurements all contain multiple lens, mirrors and filters for directing the emission towards the detector. The optical components and principles have not changed in over 100 years. A modern microscope has the same shape and configuration as those over 100 years old. In contrast there has been enormous progress in electronics where the cost per GB of memory has decreased 1 million-fold. We propose a widely applicable approach to fluorescence imaging that can provide high spatial resolution over large areas, easily up to the frame size of 35 mm film. This will be accomplished using Tamm structures which consist of multiple layers of dielectrics and a top metallic layer; which are called Tamm structures (TS). These structures contain no nanoscale features in the x-y plane and vapor deposition can produce large area structures at low cost. Tamm structures support optical modes which are perpendicular to the surface, and we have recently shown fluorophores close to the surface couple to these modes and also radiate perpendicular to the surface. In this project the Tamm structures will be placed directly onto CMOS imaging detectors (CID) for imaging. We refer to this method as coupled-emission microscopy (CEM). Practical CEM devices require improved z-axis confinement and reduced sample-to-detector distances. We propose development of CEM to obtain a spatial resolution of 1 μm or better. This will be accomplished by optical simulations and refined methods for preparation of Tamm structures and other multi- layer structures (MLS). Two independent methods will be used to test for improved z-axis confinement and improved coupling efficiency. Commercial CIDs contain protective cover slips which prevent close contact with the Tamm structures. Methods will be developed to place the TS directly onto the CID surface and for higher spatial resolution fabrication of the Tamm structure directly on the CID surface. Various methods of illumination and/or thin-film filters will be developed to reject incident light. Spatial resolution will be measured using fluorescent nanobeads (NBs) as point sources will also be used to determine the number of independent measurable locations. The effects of surface topology will be examined for effects on spatial resolution and sensitivity by increases in fluorophore-TS coupling efficiency. We will also examine alternative structures which are known to display perpendicular modes such as the three layer metal-dielectric-metal (MDM) structures and structures which do not contain any metal and display optical Tamm states (OTS). Replacement of even a small fraction of existing instruments with CEM devices would have a large impact on biology and medicine.

摘要 荧光成像技术在几乎所有的生物科学中得到了广泛的应用。示例包括以下成像 带有DNA或蛋白质斑点的显微镜载玻片，像细胞因子或 捕获免疫测定。荧光发生在所有方向上，并且有必要收集尽可能多的荧光。 尽可能地发射，并将发射聚焦到检测器上。用于这些测量的仪器都 包含多个透镜、反射镜和滤光器，用于将发射导向检测器。光学部件 原则一百多年来都没有改变现代显微镜的形状和结构 和那些超过100岁的人一样。相比之下，电子产品取得了巨大的进步， GB内存减少了100万倍。 我们提出了一种广泛适用的荧光成像方法，可以提供高空间分辨率 在大面积上，很容易达到35毫米胶片的帧尺寸。这将使用Tamm结构来完成 其由多层金属层和顶部金属层组成;其被称为Tamm结构（TS）。 这些结构在x-y平面中不包含纳米尺度特征，并且气相沉积可以产生大面积的 低成本的结构。Tamm结构支持垂直于表面的光学模式， 最近已经表明，靠近表面的荧光团与这些模式耦合，并且还垂直于 表面上在这个项目中，Tamm结构将直接放置在CMOS成像探测器（CID）上， 显像我们将这种方法称为耦合发射显微镜（CEM）。实用的CEM设备需要 改进的z轴限制和减小的样品到检测器的距离。 我们建议发展CEM以获得1 μm或更好的空间分辨率。这将是 通过光学模拟和精细的方法来制备Tamm结构和其他多- 层结构（MLS）。两种独立的方法将用于测试改进的z轴限制， 提高耦合效率。商业CID包含保护性盖玻片，可防止与 塔姆结构。将开发将TS直接放置在CID表面上的方法， 直接在CID表面上的Tamm结构的空间分辨率制造。各种照明方式 和/或将开发薄膜滤光器以拒绝入射光。空间分辨率将使用 荧光纳米珠（NB）作为点源也将用于确定独立的数量 可测量的位置。将检查表面拓扑结构对空间分辨率和 通过增加荧光团-TS耦合效率提高灵敏度。我们还将研究替代结构， 已知显示垂直模式，例如三层金属-电介质-金属（MDM）结构， 不包含任何金属并显示光学Tamm态（OTS）的结构。更换即使是一个小 现有仪器的一部分与CEM设备将对生物学和医学产生巨大影响。