Coupled Emission Microscopy for the Biosciences

摘要 荧光成像在几乎所有的生物科学中都得到了广泛的应用。例如，成像 显微镜载玻片上有DNA或蛋白质的斑点，细胞因子或细胞因子等分子类别的多重分析 捕获免疫分析。荧光发生在各个方向，有必要收集尽可能多的 尽可能地发射，并将发射聚焦于探测器。这些测量的仪器都 包含多个透镜、反射镜和滤光片，用于将发射引导到探测器。光学元件 100多年来，原则没有改变。现代显微镜具有相同的形状和配置 就像100岁以上的人一样。相比之下，电子产品取得了巨大的进步，每台设备的成本 GB内存减少了100万倍。 我们提出了一种可以提供高空间分辨率的广泛适用的荧光成像方法。 覆盖面积大，可轻松达到35 mm胶片的边框尺寸。这将使用TAMM结构来完成 它由多层介质和顶层金属层组成，称为TAMM结构(TS)。 这些结构在x-y平面上不包含纳米尺度的特征，并且气相沉积可以产生很大面积 低成本的结构。TAMM结构支持垂直于表面的光学模式，我们 最近显示了靠近地表的荧光团耦合到这些模式，并且也垂直于 表面。在这个项目中，TAMM结构将被直接放置在CMOS成像探测器(CID)上 成像。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 改进了z轴限制，缩短了样品到探测器的距离。 我们建议发展CEM以获得1μm或更高的空间分辨率。这将是 通过光学模拟和改进的方法来制备TAMM结构和其他多层结构. 层结构(MLS)。将使用两种独立的方法来测试改进的z轴限制和 提高了耦合效率。商用CID包含保护盖卡套，防止与 塔姆建筑。将开发将TS直接放置在CID表面上的方法，并针对更高 直接在CID表面制作TAMM结构的空间分辨率。各种照明方法 和/或薄膜滤光片将被开发来抑制入射光。空间分辨率将使用 荧光纳米棒(NBS)作为点源也将被用来确定独立的数量 可测量的位置。将检查表面拓扑结构对空间分辨率的影响以及 通过提高荧光团-TS偶联效率来提高灵敏度。我们还将研究其他结构，这些结构 已知显示垂直模式，例如三层金属-介质-金属(MDM)结构和 不包含任何金属的结构，并显示光学Tamm态(OTS)。换掉哪怕是一小块 现有仪器中的一小部分带有CEM设备，将对生物学和医学产生重大影响。

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.