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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.

项目概要/摘要当前疾病诊断和治疗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的国家。苏木精-伊红 (H&E) 染色样品的明场成像代表为临床组织病理学中准确、全面的评估和诊断提供了主要方法奥吉 [1, 2]。疾病表征的其他技术包括分子特异性标记，并使用免疫印迹技术。用于明场和荧光显微镜的免疫组织化学或免疫荧光技术，分别积极地。使用后者，可以同时检查多种分析物 [3, 4]。不幸的是，复杂性荧光显微镜的光学设计与多重荧光报告基因的数量成比例，以便视觉- 化，从而限制了其临床应用[5]。另一个感兴趣的领域是探索可能的临床相关信息其空间分辨率超出了传统显微镜所能达到的水平。典型荧光显微镜通常受到衍射的限制，光学分辨率约为 200 nm。尽管该决议使大细胞结构的可视化，不支持细胞器和亚细胞器水平的检查超微结构的形态变化可能与疾病相关，如神经退行性变、年龄和疾病癌症[6-10]。最近，光学超分辨率技术的出现，实现了成像分辨率分辨率优于 50 nm。然而，此类技术依赖于复杂的硬件，目前成本太高将纳入典型的临床病理学预算。电子显微镜（EM）系统也是一种可用的 ble 选项，并且通常以约 1 nm 的分辨率进行成像 – 然而，这些技术并未广泛使用，也没有非常适合分子特异性成像[11-14]。其他问题，包括尺寸、成本、有限的视野、样品制备方案的复杂性阻碍了 EM 被纳入标准临床工作中流动。该项目将开发一种稳健、相对简单且低成本的光学系统，用于分子特异性多光谱荧光成像，空间分辨率约为 70 nm，远低于经典的 200 nm 光学分辨率分辨率限制。为此，将开发计算结构照明（SI）显微镜框架使用未校准的照明模式实现超分辨率。该框架将使用单波长紫外（UV）激发，已证明能够同时激发多 ple荧光记者。这项工作的具体创新包括 SI 显微镜的新颖重新表述，在系统未知的情况下，使用计算优化来大幅提高成像分辨率和不完美之处。此外，由于基于 UV 的激发波长短了 2 倍多与荧光团的可见光发射波长相比，预计分辨率增益将大于 2 倍，从而实现亚100纳米的空间分辨率。如果成功，该项目的目标将结合以下优势多光谱光学成像具有亚 100 nm 空间分辨率的优势，可创建信息更丰富的图像是电子显微镜技术要求较低的替代品，可应用于生物学和组织病理学领域。