Cryogenic High-throughput Cellular Imaging System

项目概要/摘要 X射线显微镜已经发展成为一种重要的超微结构成像方法， 并在三维空间中测量完整的细胞。用于细胞成像的光束线 在世界各地的同步加速器设施中建造，并且该提案的共同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。的 迄今为止的方法都集中在使用“水窗”X射线， 在285至540 eV之间，并且水是透明的，但有机成分是吸收的。 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 可以成像的细胞的大小（例如，<10 µm，当许多哺乳动物细胞为10-100 µm时）。 该SBIR提案旨在开发一种能够实现高通量的3D纳米X射线显微镜 （约30分钟）直径达80 µm的低温保存细胞的3D成像， 30 nm分辨率。该系统利用了在较高（2.7 keV）能量的X射线，其可以实现比水更高的生物样品对比度 窗户X光片此外，该系统将使几个主要优势超过水 窗口X射线显微镜，包括更大的细胞成像（80µm对10µm），更大的深度， 更高的3D分辨率和实际好处（更稳定的X射线源和更大的 工作距离，以结合相关技术）。显微镜使用该公司的 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2.7 keV环境系统， 低温操作并优化其细胞成像性能。拟议的第二阶段 24-一个月的项目是开发一个完整的低温2.7 keV系统的细胞成像和 实验证明其在哺乳动物细胞上的性能。

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.