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.

概括 光活化定位显微镜（棕榈）和随机光学重建显微镜 （Storm）通过引入合理的方法彻底改变了超分辨率领域 在最终用户实验室中掌握并实施。现在，在整个学科中都感受到了它们的影响 它们的独特功能正应用于不断扩展的重要问题的范围。 近场扫描光学显微镜（NSOM）是另一种超分辨率技术 属性的完整性掌握了棕榈和风暴。 NSOM是一种使用的扫描探针技术 特殊制造的光纤问题以测量超分辨率荧光和样品地形， 相似地。这在细胞形状和形态学的生物科学中特别有用 可以将特征直接与荧光图像中特异性标记的物种进行比较。迄今为止， 但是，NSOM在生物科学中的影响很小。这主要是由于 繁重的实施要求和光纤问题的性能不佳。克服 这些挑战，我们提出了一种全新的方法，将光学对比机制集成 原子力显微镜（AFM）。 扫描谐振器显微镜（SRM）使用附着在 常规的AFM探针用于超分辨率成像。该方法利用耳语画廊模式 （WGM）在连接的谐振器中激发的共振，以了解感官或令人兴奋的样本属性。与众不同 常规的NSOM，问题很容易在解剖显微镜下组装，而SRM需要 仅对商业AFM平台的最小修改。这应该在宽度上采用 最终用户实验室。我们已经开发了一个原型SRM，并通过 类似地量化具有超分辨率的薄膜的样品折射率和地形。这 这里的总体目标是开发能够简单荧光，折射的下一代SRM 索引和复杂生物样品的地形测量。 此处提出的研究计划将：（目标1）为SRM TIP开发一个“沿”光纤接头 兴奋，可以在厚的生物样品和（AIM 2）测试上实现超分辨率荧光成像， 通过研究膜联蛋白VI定位，在实际生物系统上验证和基准SRM性能 钙刺激后，在固定的人动脉平滑肌细胞中。成功完成 工作将引入一个新的超分辨率工具，该工具可以在最终用户实验室中轻松采用，并具有独特的 现有技术的功能完善。对于未来，很容易设想额外的敏感性 通过特定涂料或光学谐振器的修改将功能与SRM集成 尖端末端。