Scanning Resonator Microscopy

扫描谐振显微镜

• 批准号: 9954076
• 负责人: Robert C. Dunn
• 金额: $ 7.1万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9954076
• 关键词: Adopted; Adoption; Annexin A6; Atomic Force Microscopy; Award; Benchmarking; Binding Proteins; Biological; Biological Process; Biological Sciences; Calcium; Cell Shape; Cell membrane; Cellular Morphology; Charge; Chemistry; Complex; Development; Dimensions; Discipline; F-Actin; Fiber Optics; Film; Fluorescence; Fluorescence Microscopy; Fluorescent Probes; Future; Goals; Human; Image; Label; Laboratories; Light; Lipids; Measurement; Measures; Membrane Microdomains; Methods; Microscope; Microscopy; Microspheres; Modification; Monitor; Nobel Prize; Optics; Performance; Procedures; Property; Publications; Refractive Indices; Research; Resolution; Sampling; Scanning; Smooth Muscle Myocytes; Source; Surface; Techniques; Technology; Testing; Thick; Thinness; Time; Work; base; biological systems; fluorescence imaging; next generation; novel strategies; prototype; reconstruction; single molecule; tool

Summary Photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) have revolutionized the super-resolution field by introducing approaches that are reasonably mastered and implemented in the end-user laboratory. Their impact is now felt across the disciplines where their unique capabilities are being applied to an ever-expanding spectrum of important problems. Near-field scanning optical microscopy (NSOM) is another super-resolution technique that has attributes complementary to those of PALM and STORM. NSOM is a scanning probe technique that uses specially fabricated fiber optic probes to measure super-resolution fluorescence and sample topography, simultaneously. This is particularly useful in the biological sciences, where cell shape and morphological features can be compared directly with species specifically labeled in the fluorescence image. To date, however, the impact of NSOM in the biological sciences has been modest. This is mainly due to burdensome implementation requirements and poor performance of the fiber optic probes. To overcome these challenges, we propose a completely new approach for integrating optical contrast mechanisms with atomic force microscopy (AFM). Scanning resonator microscopy (SRM) uses a small dielectric microsphere attached at the end of conventional AFM probe for super-resolution imaging. The approach exploits whispering gallery mode (WGM) resonances excited in the attached resonator to sense or excite sample properties. Unlike conventional NSOM, the probes are easily assembled under a dissecting microscope and SRM requires only minimal modifications to commercial AFM platforms. This should enable widespread adoption in the end user lab. We have developed a prototype SRM and demonstrated the feasibility of this approach by simultaneously quantifying sample refractive index and topography of thin films with super-resolution. The overall goal here is to develop the next generation SRM capable of simultaneous fluorescence, refractive index, and topography measurements on complex biological samples. The research plan proposed here will: (Aim 1) develop a “ride along” fiber optic coupler for SRM tip excitation that enables super-resolution fluorescence imaging on thick biological samples and (Aim 2) test, validate, and benchmark SRM performance on a real biological system by studying annexin VI localization in fixed human arterial smooth muscle cells following calcium stimulation. The successful completion of this work will introduce a new super-resolution tool that can easily be adopted in the end user lab, with unique capabilities complementary to existing technologies. For the future, it is easy to envision additional sensing capabilities being integrated with SRM through specific coatings or modifications of the optical resonator at the tip end.

摘要 光活化定位显微镜(Palm)和随机光学重建显微镜 (STORM)通过引入合理的 在最终用户实验室中掌握并实施。他们的影响现在可以在各个学科中感受到 它们独特的能力正被应用于一系列不断扩大的重要问题。 近场扫描光学显微镜(NSOM)是另一种超分辨率技术，具有 与Palm和Storm的属性互补。NSOM是一种扫描探头技术，使用 专门制造的光纤探头，用于测量超分辨率荧光和样品形貌， 同时。这在生物科学中特别有用，在生物科学中，细胞的形状和形态 特征可以直接与荧光图像中专门标记的物种进行比较。到目前为止， 然而，NSOM在生物科学方面的影响并不大。这主要是因为 光纤探头实施要求繁重，性能较差。要克服 针对这些挑战，我们提出了一种全新的方法，将光学对比机制与 原子力显微镜(AFM)。 扫描谐振器显微镜(SRM)使用一个小的介电微球附着在 用于超分辨率成像的常规AFM探头。该方法利用了耳语画廊模式 在连接的谐振器中激发的(WGM)共振，以检测或激发样品的特性。不像 传统的NSOM，探针很容易在解剖显微镜下组装，而SRM需要 只需对商用AFM平台进行极小的修改。这应该能够在 最终用户实验室。我们已经开发了一个SRM原型，并通过以下方式演示了该方法的可行性 用超分辨率同时定量样品折射率和薄膜的形貌。这个 这里的总体目标是开发能够同时荧光、折射的下一代SRM 指数，以及复杂生物样品的地形测量。 本文提出的研究计划将：(目标1)研制一种用于固体火箭发动机叶尖的顺行光纤耦合器。 能够在厚生物样品上进行超分辨率荧光成像的激发和(AIM 2)测试， 通过研究Annexin VI本地化在真实生物系统上验证和基准SRM性能 在钙刺激后固定的人动脉平滑肌细胞中。成功地完成这项工作 Work将推出一种新的超分辨率工具，该工具可以很容易地在最终用户实验室中采用，具有独特的 补充现有技术的能力。对于未来，很容易想象更多的传感 通过光学谐振器的特定涂层或修改与SRM集成的功能，请参见 小费结束了。