Cryo ptychography combined with x-ray fluorescence analysis of metals in cells

细胞中金属的冷冻层析术与 X 射线荧光分析相结合

• 批准号: 8998032
• 负责人: Chris Johnson Jacobsen
• 金额: $ 28.49万
• 依托单位: NORTHWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-02-15 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8998032
• 关键词: Biological; Biological Preservation; Biomedical Research; Breast; Cell Culture Techniques; Cells; Cellular Structures; DNA; Development; Disease; Electron Microscope; Electrons; Elements; Fluorescence; Fluorescence Microscopy; Formulation; Freezing; Funding; Gadolinium; Goals; Health; Human; Image; Investments; Ions; Label; Life; Light; Malignant Neoplasms; Measurement; Metals; Methods; Microscope; Microscopy; Microtomy; Mitochondria; Morphologic artifacts; N.I.H. Research Support; Optics; Organelles; Pharmaceutical Preparations; Phase; Photons; Preparation; Proteins; Protocols documentation; Radiation; Research; Research Personnel; Research Project Grants; Resolution; Roentgen Rays; Role; Route; Sampling; Scanning; Source; Specimen; Synchrotrons; Thick; Time; Tissues; Titania; Titanium; Trace Elements; Trace metal; United States National Institutes of Health; Variant; Water; Work; Zinc Fingers; base; beamline; biological systems; blind; cancer therapy; contrast imaging; cryogenics; design; fluorescence imaging; fluorescence microscope; follow-up; gadolinium oxide; imaging modality; instrument; lens; member; nanomaterials; nanoparticle; novel; operation; programs; research and development

DESCRIPTION (provided by applicant): X-ray fluorescence microscopes offer the highest sensitivity for studies of the role of trace metals in cells, and they provide essential informatio for understanding the ultrastructural targeting of nanoparticles used for potential cancer therapies. With ARRA support from the NIH, co-PI Woloschak et al. have obtained the Bionanoprobe at Argonne's Advanced Photon Source (the nation's premier hard x-ray synchrotron light source facility). This is a first-of-its-kind instrument for x-ray fluorescence studies of trace elements in cells and tissue sections which offers a thousandfold improvement in sensitivity compared to electron microprobes, cryo transfer conditions to work with fully hydrated specimens at the highest preparation fidelity possible, and 3D tomographic imaging capabilities. Our proposal is to devote the scientific and technical effort needed to realize this new instrument's potential. Since hard x-ray microscopes are able to image samples with thicknesses ranging up to tens of micrometers, we can study whole cells and tissue sections in 3D in a way that electron microscopes cannot, and with a combination of sub-50 nm spatial resolution and sensitive trace element quantitation available in no other method. Cryogenic conditions are required to minimize radiation damage effects and maximize the fidelity of ultrastructure and trace element concentrations, so we need to develop and validate the novel cryogenic sample preparation methods appropriate for our unique sample types. X-ray fluorescence is relatively blind to the light elements (such as organic materials and water) that comprise nearly all the mass and ultrastructure of cells. Fortunately, we have pioneered several methods that can make visible what was once "dark," and we will develop a variant of these approaches (ptychography, a method of scanned coherent imaging that delivers quantitative phase contrast and spatial resolution beyond the limit of x-ray optics) and turn this into a routin, simultaneous-with-fluorescence imaging method for users of the Bionanoprobe. To validate these approaches and work from the beginning on a crucial biomedical research project, we will do this in the context of ongoing research in the use of DNA-conjugated nanoparticles containing titanium and/or gadolinium that are meant to target mitochondria for the treatment and imaging of prostrate, breast, and other cancers. In this way, we will develop the methods needed to fully realize the investment NIH has already made in the Bionanoprobe.

描述（由申请人提供）：x射线荧光显微镜为研究细胞中微量金属的作用提供了最高的灵敏度，它们为了解用于潜在癌症治疗的纳米颗粒的超微结构靶向提供了必要的信息。在美国国立卫生研究院ARRA的支持下，Woloschak等人在阿贡的先进光子源（美国首屈一指的硬x射线同步加速器光源设施）获得了Bionanoprobe。这是一种用于细胞和组织切片中微量元素的x射线荧光研究的首创仪器，与电子显微探针相比，它的灵敏度提高了千倍，在最高制备保真度的条件下，低温转移条件可以与完全水化的标本一起工作，并具有3D层析成像能力。我们的建议是投入必要的科学和技术努力来实现这一新仪器的潜力。由于硬x射线显微镜能够对厚度达数十微米的样品进行成像，因此我们可以以电子显微镜无法实现的方式以3D方式研究整个细胞和组织切片，并且结合了低于50纳米的空间分辨率和灵敏的微量元素定量，这是其他方法无法实现的。低温条件需要最大限度地减少辐射损伤效应，最大限度地提高超微结构和微量元素浓度的保真度，因此我们需要开发和验证适合我们独特样品类型的新型低温样品制备方法。x射线荧光对构成细胞几乎所有质量和超微结构的轻元素（如有机物质和水）相对不可见。幸运的是，我们已经开创了几种方法，可以使曾经的“黑暗”变得可见，我们将开发这些方法的一种变体（ptychography，一种扫描相干成像方法，提供定量相位对比和超越x射线光学极限的空间分辨率），并将其转变为常规的，与荧光同时成像的方法，为Bionanoprobe的用户提供。为了验证这些方法，并从一项重要的生物医学研究项目开始，我们将在正在进行的使用含有钛和/或钆的dna共轭纳米颗粒的研究背景下进行这项研究，这些纳米颗粒旨在靶向线粒体，用于前列腺癌、乳腺癌和其他癌症的治疗和成像。通过这种方式，我们将开发出充分实现NIH已经对Bionanoprobe进行的投资所需的方法。