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

• 批准号: 9788760
• 负责人: Shwetadwip Chowdhury
• 金额: $ 6.37万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 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
• 来源: https://reporter.nih.gov/project-details/9788760
• 关键词: Age; Algorithms; Area; Biology; Biomedical Engineering; Biopsy; Brightfield Microscopy; Budgets; Caliber; California; Cellular Structures; Chromatin; Clinical; Clinical Pathology; Complex; Computational algorithm; Custom; Development; Diagnosis; Diagnostic; Disease; Disease Management; Electron Microscopy; Elements; Evaluation; Fluorescence; Fluorescence Microscopy; Fluorescent Antibody Technique; Foot Process; Formalin; Glutaral; Hematoxylin and Eosin Staining Method; Histology; Histopathology; Image; Imagery; Immunohistochemistry; Label; Laboratories; Light; Lighting; Malignant Neoplasms; Methods; Microscope; Microscopy; Microspheres; Mitochondria; Molecular; Morphology; Nature; Nerve Degeneration; Nuclear; Optics; Organelles; Pathology; Pattern; Performance; Positioning Attribute; Protocols documentation; Recovery; Renal carcinoma; Reporter; Resistance; Resolution; Sampling; Specimen; Stains; Structure; Sulfur; Surface; System; Techniques; Technology; Tern; Time; Tissues; Translating; Translations; Uncertainty; Universities; Validation; Variant; Visual; Work; base; clinical practice; clinically relevant; comparative; computer framework; cost; design; enhancing factor; fluorescence imaging; fluorescence microscope; fluorophore; histopathological examination; image reconstruction; imaging system; improved; innovation; interest; lens; light microscopy; nanoscale; new technology; novel; off-label use; optical imaging; optical spectra; prevent; prototype; reconstruction; research clinical testing; routine imaging; sample fixation; simulation; transmission process; ultraviolet; virtual

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.

项目摘要/摘要 疾病诊断和管理的当前临床实践通常依赖于组织病理学检查 - 通过光学显微镜的组织国家。苏木精 - 欧洲蛋白（H＆E）染色的样品的明亮菲尔德成像 发送主要的方法，以进行准确，全面的评估和诊断，以 OGY [1，2]。疾病表征的其他技术涉及分子特异性标记，并使用IM- Muno组织化学或免疫荧光技术，用于明亮场和荧光显微镜，分解 蒂。使用后者，可以简单地检查多个分析物[3，4]。不幸的是，复杂性 荧光显微镜的光学设计尺度，具有多重荧光记者的数量 ize，因此限制了其临床实用性[5]。感兴趣的另一个领域是探索可能与临床相关的信息 在空间分辨率上存在于传统显微镜可以实现的范围之外。典型的荧光 显微镜通常通过衍射限制为〜200 nm的光学分辨率。虽然该决议启用了 大型细胞结构的可视化，它不支持检验细胞器和亚轨道级 形态变化可能与疾病相关的超微结构，如神经退行性，年龄和 癌症[6-10]。最近，已经引入了光学超分辨率技术，以实现成像的重点 小超过50 nm。但是，此类技术依赖复杂的硬件，目前太昂贵了 将其纳入典型的临床病理预算中。电子显微镜（EM）系统也是可用的 ble选项，并在〜1 nm的分辨率上进行例行图像 - 但是，这些都不可用，也不是 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PREP方案的复杂性已阻止EM被纳入标准临床工作中 - 流动。该项目将开发出一个可靠的，相对简单且低成本的光学系统，用于分子特异性 〜70 nm的空间分辨率下的多光谱荧光成像，良好 分辨率限制。为此，将开发用于计算结构化照明（SI）显微镜的框架 使用未校准的照明模式实现超分辨率。该框架将使用单个框架部署 波长紫外线（UV）兴奋，这表明了同时兴奋的能力 PLE荧光记者。这项工作中的特定创新包括新的SI显微镜改革， 使用计算优化在系统未知的存在下可靠地增加成像分辨率 和缺陷。此外，因为基于紫外线的兴奋的波长超过2倍 与荧光团的可见发射波长相比，通过预期的因素分辨率提高到2个， 因此实现了以下的100 nm空间分辨率。如果成功，该项目的目标将结合利益 多光谱光学成像具有亚100 nm空间分辨率的优势，以创建更有信息 在生物学和组织病理学范围内应用，对电子显微镜的要求较少。