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

摘要 X射线荧光显微镜(XFM)为研究痕量金属的作用提供了最高的灵敏度 细胞，它们为理解纳米颗粒的超微结构靶向提供了必要的信息。 用于潜在的癌症治疗。在NIH的支持下，我们已经开发出快速冷冻细胞的方法 为了最好地保存微量金属含量以及细胞超微结构，并表明低温X- 射线荧光显微镜可以有效地减轻辐射损伤的限制 BionanoProbe，一种在Argonne的高级光子源(APS)运行的仪器，向 研究人员基于同行评审的免费波束时间提案。为了补充XFM的能力， 为了定量分析图像中痕量元素的分布，我们开发了高通量x射线照相技术(a 扫描相干束成像方法)超越透镜限制，同时获得18 nm分辨率 冷冻水合真核细胞的图像，通过提供细胞的高分辨率视图来补充XFM 超微结构。我们建议开发和验证BionanoProbe的低温共聚焦光学显微镜- 安装样品以补充XFM成像选择性标记的蛋白质的能力，并移动 从2D成像到3D成像的印刷术。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使用DNA偶联 含有钛和/或Gd的纳米颗粒，旨在靶向治疗线粒体和 对前列腺癌、乳腺癌和其他癌症的成像。通过这种方式，我们将开发所需的方法来充分实现 NIH已经对BionanoProbe进行了投资，并建立在Argonne对增加 它在APS的一个新实验站的可用访问时间。

Summary X-ray fluorescence microscopes (XFM) offer the highest sensitivity for studies of the role of trace metals in cells, and they provide essential information for understanding the ultrastructural targeting of nanoparticles used for potential cancer therapies. With NIH support, we have developed approaches to rapidly freeze cells for the best preservation of trace metal content as well as cellular ultrastructure, and have shown that cryo x- ray fluorescence microscopy can be used to effectively mitigate radiation damage limitations in the Bionanoprobe, an instrument operated at the Advanced Photon Source (APS) at Argonne and open to researchers based on peer-reviewed, no-cost beamtime proposals. In order to complement XFM's ability to quantitatively image trace element distributions, we have developed high throughput x-ray ptychography (a scanned coherent beam imaging method) to go beyond lens limits and simultaneously obtain 18 nm resolution images of frozen hydrated eukaryotic cells, complementing XFM by providing a high resolution view of cellular ultrastructure. We propose here to develop and validate cryo confocal light microscopy of Bionanoprobe- mounted samples to complement XFM with the capability to image selectively labeled proteins, and to move ptychography from 2D to 3D imaging. 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, and build upon Argonne's investment in increasing its available access time at a new experimental station at the APS.