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

基本信息

批准号：
8620670
负责人：
Chris Johnson Jacobsen
金额：
$ 28.57万
依托单位：
NORTHWESTERN UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-02-15 至 2017-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8620670
关键词：
Biological Biological Preservation Biomedical Research Breast Cell Culture Techniques Cells Cellular Structures DNA Development Disease Drug Formulations Electron Microscope Electrons Elements Fluorescence Fluorescence Microscopy Freezing Funding Gadolinium Goals 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 cryogenics design fluorescence imaging fluorescence microscope follow-up gadolinium oxide imaging modality instrument lens member nanomaterials nanoparticle novel operation programs public health relevance research and development

项目摘要

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射线荧光显微镜为痕量金属在细胞中的作用的研究提供了最高的敏感性，它们为理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不可少的信息。在NIH的ARRA支持下，Co-Pi Woloschak等。已经在Argonne的高级光子源（该国主要的硬X射线同步器光源设施）中获得了Bionanopre。这是一种针对细胞和组织切片中微量元素X射线荧光研究研究的首个仪器，与电子微探针相比，敏感性提高了千倍的敏感性，冷冻条件，可在可能的最高制备屈曲中与完全水合的样品一起使用，并具有最高的制备性能和3D断层造影成像能力。我们的建议是投入实现这种新工具潜力所需的科学和技术努力。由于硬X射线显微镜能够成像具有厚度范围为数十微米的厚度的样品，因此我们可以以3D的方式研究全细胞和组织切片，以使电子显微镜无法无法进行，并且与其他方法无需其他方法中可用的50 nm空间分辨率和敏感的微量元素定量。需要低温条件以最大程度地减少辐射损伤效应并最大程度地提高超微结构和痕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射线荧光对几乎构成细胞的质量和超微结构的光元素（例如有机材料和水）相对盲目视而不见。 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.为了验证这些方法和工作从一开始的关键生物医学研究项目，我们将在不断研究的背景下使用含有钛和/或Gadolinium的DNA偶联的纳米颗粒，旨在针对线粒体的治疗方法，以治疗和成像提倡，乳房和其他癌症。这样，我们将开发完全意识到NIH已经在Bionanoprobe中进行的投资所需的方法。