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Structured Illumination Computational Microscopy with UV Surface Excitation (MUSE) for Multispectral Super-Resolution Histology

用于多光谱超分辨率组织学的紫外表面激发 (MUSE) 结构照明计算显微镜

基本信息

批准号：
10213544
负责人：
Shwetadwip Chowdhury
金额：
$ 2.31万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-01 至 2020-12-31
项目状态：
已结题

项目摘要

Project Summary/Abstract Current clinical practices for the diagnosis and management of diseases often rely on histopathological exami- nation of tissue via optical microscopy. Brightfield imaging of hematoxylin-eosin (H&E)-stained samples repre- sents the predominant approach for accurate and comprehensive evaluation and diagnosis in clinical histopathol- ogy [1, 2]. Additional techniques for disease characterization involve molecularly specific labeling, and use im- munohistochemical or immunofluorescence techniques for brightfield and fluorescence microscopy, respec- tively. Using the latter, multiple analytes can be examined simultaneously [3, 4]. Unfortunately, the complexity of a fluorescent microscope’s optical design scales with the number of multiplexed fluorescent reporters to visual- ize, thus limiting its clinical utility [5]. Another area of interest is to explore clinically relevant information that may exist at spatial resolutions beyond what can be achieved with conventional microscopes. Typical fluorescence microscopy is generally limited by diffraction to an optical resolution of ~200 nm. Though this resolution enables visualization of large cellular structures, it does not support examination of organelle- and suborganelle-level ultrastructure whose morphological changes can correlate with disease, as seen in neurodegeneration, age, and cancer [6-10]. Recently, optical super-resolution technologies have been introduced that achieve imaging reso- lutions better than 50 nm. However, such technologies depend on complex hardware and are currently too costly to be incorporated into typical clinical pathology budgets. Electron microscopy (EM) systems are also an availa- ble option, and routinely image at resolutions of ~1 nm – however, these are not widely available and are not well suited for molecular specific imaging [11-14]. Additional issues, including size, cost, limited field-of-view, and complexity of sample-prep protocols have prevented EM from being incorporated into standard clinical work- flow. This project will develop a robust, comparatively simple, and low-cost optical system for molecularly-specific multispectral fluorescence imaging at spatial resolutions of ~70 nm, well beneath the classical 200 nm optical resolution limit. To do so, a framework for computational structured illumination (SI) microscopy will be developed to enable super-resolution using uncalibrated illumination patterns. This framework will be deployed using single- wavelength ultraviolet (UV) excitation, which has demonstrated capabilities for simultaneous excitation of multi- ple fluorescent reporters. Specific innovations in this work include a novel reformulation of SI microscopy that uses computational optimization to robustly increase imaging resolution in the presence of system unknowns and imperfections. Furthermore, because UV-based excitation has wavelengths more than a factor of 2 shorter than the fluorophores’ visible emission wavelengths, resolution gains by factors greater than 2 are expected, hence enabling sub-100-nm spatial resolutions. If successful, the aims of this project will combine the benefits of multispectral optical imaging with the advantages of sub-100-nm spatial resolution to create a more informative and less demanding alternative to electron microscopy, with applications across biology and histopathology.

项目总结/摘要目前用于疾病诊断和管理的临床实践通常依赖于组织病理学检查，通过光学显微镜观察组织。苏木精-伊红（H&E）染色样本的双视野成像代表了指出了在临床组织病理学中准确和全面评价和诊断的主要方法， ogy [1，2].用于疾病表征的其他技术包括分子特异性标记，并使用免疫组织化学（IM）。免疫组织化学或免疫荧光技术，分别用于明视野和荧光显微镜，很好使用后者，可以同时检查多个分析物[3，4]。不幸的是，荧光显微镜的光学设计与多路荧光报告器的数量成比例，因此限制了其临床应用[5]。另一个感兴趣的领域是探索临床相关信息，其空间分辨率超过了传统显微镜所能达到的分辨率。典型荧光显微术通常被衍射限制到~200 nm的光学分辨率。虽然这项决议使大细胞结构的可视化，它不支持细胞器和亚细胞器水平的检查超微结构，其形态学变化与疾病相关，如神经变性、年龄和癌症[6-10]。最近，已经引入了光学超分辨率技术，其实现成像分辨率。溶液优于50纳米。然而，这类技术依赖于复杂的硬件，目前成本太高纳入典型的临床病理学预算。电子显微镜（EM）系统也是一个有用的，可选，并以约1 nm分辨率常规成像-然而，这些并不广泛可用，非常适合分子特异性成像[11-14]。其他问题，包括尺寸、成本、有限的视场，和复杂的样品制备方案，阻碍了EM被纳入标准的临床工作- 流该项目将开发一个强大的，相对简单的，低成本的光学系统，用于分子特异性多光谱荧光成像的空间分辨率约为70 nm，远低于经典的200 nm光学分辨率极限为此，将开发计算结构照明（SI）显微镜的框架以使用未校准的照明图案实现超分辨率。该框架将使用单- 波长紫外（UV）激发，它已被证明能够同时激发多个双荧光报告基因。这项工作的具体创新包括一种新的SI显微镜的重新表述，在系统未知的情况下，使用计算优化来稳健地增加成像分辨率和缺陷。此外，因为基于UV的激发具有比2倍更短的波长比荧光团的可见发射波长，预期分辨率增益大于2倍，因此能够实现亚100纳米的空间分辨率。如果成功，这个项目的目标将联合收割机的好处具有亚100纳米空间分辨率优势的多光谱光学成像，可创建信息更丰富的以及要求较低的电子显微镜替代品，应用于生物学和组织病理学领域。