Cryogenic High-throughput Cellular Imaging System

项目摘要/摘要 X射线显微镜已经发展成为可视化的一种重要的超微结构成像方法 并测量三维的完整细胞。专用于细胞成像的光束线具有 在世界各地的同步加速器设施中建造，以及这项提议的共同PI 开发了第一台用于细胞成像的商用实验室X射线显微镜。这个 到目前为止，方法都是围绕着“水之窗”x射线的使用，具有能量 在285到540 eV之间，水是透明的，但有机含量正在吸收。 然而，使用这些低能量x射线有几个主要缺点：它们严重限制了 可成像的细胞大小(例如，当许多哺乳动物细胞为10-100微米时，为10微米)。 这项SBIR建议旨在开发一种能够高通量的3D纳米x射线显微镜 (~30分钟)对直径最大为80微米的低温保存细胞进行3D成像 30 Nm分辨率。该系统利用了较高(2.7)的Zernike相位对比度现象 Kev)能量x射线，生物样品的对比度甚至比水更高 窗口X光片。此外，与水相比，该系统将具有几个主要优势 窗口X射线显微镜，包括更大的细胞成像(80微米对10微米)，更大的深度 用于更高3D分辨率的领域和实际好处(更稳定的X射线源和更大的 工作距离以纳入相关技术)。显微镜使用的是该公司的 获得专利的高亮度X射线源和专有的X射线光学技术。 该项目将开发提议公司现有的2.7kev环境系统，以实现 低温操作，并优化其细胞成像性能。建议的第二期工程 为期24个月的项目是开发一个完整的2.7kev低温细胞成像系统，并 在哺乳动物细胞上实验演示了它的性能。

Project Summary/Abstract X-ray microscopy has evolved into an important ultrastructure imaging approach for visualizing and measuring intact cells in three dimensions. Beamlines dedicated to cellular imaging have been constructed at synchrotron facilities around the world, and the co-PI of this proposal developed the first commercial laboratory x-ray microscope for cellular imaging. The approaches thus far have centered around the use of “water window” x-rays, having energies between 285 to 540eV and for which water is transparent but organic content is absorbing. However, there are several major drawbacks to using these low energy x-rays: they severely limit the size of cells that can be imaged (e.g., <10 µm when many mammalian cells are 10-100 µm). This SBIR proposal aims to develop a 3D nano x-ray microscope capable of high throughput (~30 minute) 3D imaging of cryogenically preserved cells of up to 80 µm in diameter at down to 30nm resolution. The system utilizes the phenomenon of Zernike phase contrast at higher (2.7 keV) energy x-rays, which can achieve even higher contrast for biological samples than water window x-rays. Additionally, the system will enable several major advantages over water window x-ray microscopy, including much larger cell imaging (80µm vs. 10µm), larger depth-of- field for higher 3D resolution, and practical benefits (more stable x-ray source and larger working distance to incorporate correlative techniques). The microscope uses the company’s patented high brightness x-ray source and proprietary x-ray optic technology. The project will develop the proposing company’s existing 2.7 keV ambient system to enable cryogenic operation and optimize its performance for cellular imaging. The proposed Phase II 24-month project is to develop a complete cryogenic 2.7 keV system for cellular imaging and to experimentally demonstrate its performance on mammalian cells.