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

描述(申请人提供)：X射线荧光显微镜为研究痕量金属在细胞中的作用提供了最高的灵敏度，并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要的信息。在美国国立卫生研究院的ARRA支持下，合著者Pi Woloschak等人。已经在Argonne的高级光子源(国家首屈一指的硬X射线同步辐射光源设施)获得了BionanoProbe。这是第一台用于细胞和组织切片中微量元素的X射线荧光研究的仪器，与电子探针相比，它的灵敏度提高了1000倍，低温转移条件可以在尽可能高的制备保真度下处理完全水合的样品，并且具有3D断层成像功能。我们的建议是致力于实现这一新仪器潜力所需的科学和技术努力。由于硬X射线显微镜能够对厚度高达数十微米的样品进行成像，我们可以以电子显微镜无法做到的方式研究整个细胞和组织切片，并结合其他方法无法提供的低于50 nm的空间分辨率和灵敏的微量元素定量。为了最大限度地减少辐射损伤影响并最大限度地提高超微结构和微量元素浓度的保真度，需要低温条件，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(如有机材料和水)相对看不见。幸运的是，我们已经开创了几种方法，可以让曾经的“黑暗”变得可见，我们将开发这些方法的变种(层析成像，一种扫描相干成像的方法，提供超出X射线光学限制的定量相位对比度和空间分辨率)，并将其转变为BionanoProbe用户的常规同步荧光成像方法。为了验证这些方法并从一开始就致力于一个关键的生物医学研究项目，我们将在正在进行的研究背景下进行这项工作，使用包含钛和/或Gd的DNA连接纳米颗粒，这些纳米颗粒旨在靶向线粒体，用于治疗和成像前列腺癌、乳腺癌和其他癌症。通过这种方式，我们将开发所需的方法，以充分实现NIH已经在生物探测器上进行的投资。

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.