Visualizing Live Cell Physiology with High Resolution Using Phase-Contrast STEM

使用相差 STEM 以高分辨率可视化活细胞生理学

基本信息

批准号：
10224280
负责人：
GREGORY LOUIS TIMP
金额：
$ 47.63万
依托单位：
UNIVERSITY OF NOTRE DAME
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-08-01 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10224280
关键词：
3-Dimensional Acceleration Adenosine Triphosphate Adopted Affect Area Bacteria Bacteriophage P1 Biological Biological Assay Biology Biomedical Research Cell physiology Cells Contracts Development Dose Electron Beam Electron Microscopy Electrons Elements Escherichia coli Exposure to Image Impairment Infection Letters Light Liquid substance Measures Membrane Methodology Methods Mycoplasma Nanostructures Noise Optics Outcome Phase Phototoxicity Physiological Physiology Plasmids Play Process Prokaryotic Cells Proteins Reporter Resolution Role Sampling Scanning Series Signal Transduction Silicon Slice Source Specific qualifier value Specimen Stains Streptavidin Structure Technology Temperature Testing Thick Thinness Time Titan Transmission Electron Microscopy Vacuum Water base biological systems cryogenics detector electric field graphene high resolution imaging imaging modality improved inorganic phosphate irradiation light microscopy microscopic imaging rib bone structure silicon nitride simulation technology research and development tomography uranyl acetate vector voltage

项目摘要

Project Summary This proposal describes a plan to develop a method for visualizing live cell physiology with high resolution using integrated Differential Phase Contrast-Scanning Transmission Electron Microscopy (iDPC-STEM) at low dose to promote viability. Visualizing physiology demands spatial resolution with a commensurate depth-of- field on the scale of the protein machinery (3-7 nm) that drives it without concomitant damage. With the introduction of a liquid flow cell containing water in a vacuum-tight envelope made from membranes that are transparent to the electron beam, it should be possible to scrutinize biology with high-resolution under physiological conditions with STEM. This proposal focuses on three specific technical challenges, testing solutions in a crucible of well characterized biological systems: 1. Improve resolution using a liquid flow cell formed from ultra-thin membranes and thin spacers. To reduce scattering in the membrane and liquid, it is practical to shrink the silicon nitride (SiN) membranes forming the liquid cell to 8-10 nm, and space them 150 nm apart without compromising the window integrity. To eliminate bulging in a liquid cell loaded with fluid, the windows will be reinforced with thick ribs so that a large >400 m2 area can be spanned. However, even 10 nm SiN membranes are still too thick for high-resolution imaging. So, (3 nm) thin amorphous silicon (a-Si) and atomically thin graphene or h-BN membranes spanning ribs formed from SiN will be used as windows for high-resolution imaging. The resolution will be tested using a Titan STEM by visualizing adenosine triphosphate (ATP) and fluorescent streptavidin (STR). 2. Improve contrast with iDPC-STEM imaging. To increase the visibility of transparent biological samples, a phase-contrast method for imaging, iDPC-STEM, will be adopted that uses a four-quadrant (segmented) split- detector to measure the gradient of a phase object. iDPC-STEM boasts a higher signal to noise ratio compared to conventional STEM, which offers the possibility for extremely low-dose imaging. The resolution, contrast and concomitant damage will be tested in an aberration-corrected, iDPC-equipped Themis Z (with 60 pm resolution) by visualizing ATP and fluorescent STR in thin (0-50 nm thick) liquid layers. 3. Finally, low-dose iDPC-STEM will be used with an ultra-thin liquid flow cell to visualize the smallest prokaryotic cells. If the electron probe interacts with a cell at the top membrane in the liquid cell, high- resolution images may be captured this way. Because the probe is so shallow along the optic-axis, a focus series may also be used to section a cell for 3D tomography. To test these ideas, four strains of Mycoplasma (100 nm in size) will be cultured in a shallow (150 nm) flow cell and visualized with iDPC-STEM to discover the role their nanostructure plays in infection. In specimens this thick, multi-slice simulations may be required to inform on the structure. After exposure to the beam, a LIVE/DEAD assay, along with Mycoplasma transformed with plasmids that produce an inducible fluorescent reporter will be used to score viability.

项目概要该提案描述了开发一种高分辨率可视化活细胞生理学方法的计划使用集成微分相衬扫描透射电子显微镜 (iDPC-STEM) 在低促进活力的剂量可视化需要具有相应深度的空间分辨率。蛋白质机器规模（3-7 nm）的领域驱动它而不伴随损坏。引入液体流通池，该液体流通池包含真空密封的水封套，该水封套由膜制成对电子束透明，应该可以在高分辨率下检查生物学 STEM 的生理条件该提案重点关注三个具体的技术挑战：测试。具有良好特征的生物系统的坩埚中的解决方案： 1. 使用由超薄膜和薄垫片形成的液体流通池提高分辨率。减少膜和液体中的散射，缩小氮化硅（SiN）膜是实用的将液体单元形成为 8-10 nm，并将它们间隔 150 nm，而不会影响窗口的完整性。为了消除装有液体的液体单元中的膨胀，窗户将用厚肋加固，以便大可以跨越 >400 μm2 的面积，但是，即使是 10 nm SiN 膜对于高分辨率来说仍然太厚。因此，（3 nm）薄非晶硅（a-Si）和原子薄石墨烯或 h-BN 膜跨越。由 SiN 形成的肋将用作高分辨率成像的窗口。分辨率将使用 Titan STEM 通过可视化三磷酸腺苷 (ATP) 和荧光链霉亲和素 (STR)。 2. 提高 iDPC-STEM 成像的对比度为了提高透明生物样品的可见度。将采用相衬成像方法 iDPC-STEM，该方法使用四象限（分段）分裂相比之下，iDPC-STEM 具有更高的信噪比。与传统的 STEM 相比，后者提供了极低剂量成像的可能性。伴随的损坏将在像差校正、配备 iDPC 的 Themis Z 中进行测试（下午 60 点）分辨率），通过可视化薄（0-50 nm 厚）液体层中的 ATP 和荧光 STR。 3. 最后，低剂量 iDPC-STEM 将与超薄液体流动池一起使用，以可视化最小的如果电子探针与液体细胞顶部膜上的细胞相互作用，则高- 由于探头沿光轴的距离很浅，因此可以通过这种方式捕获分辨率图像。系列也可用于对细胞进行 3D 断层扫描。为了测试这些想法，四种支原体菌株。（大小为 100 nm）将在浅层（150 nm）流动池中培养，并使用 iDPC-STEM 进行可视化以发现在样本中，可能需要这种厚的、多切片的模拟来了解它们的纳米结构在感染中的作用。暴露于光束后，进行活/死测定，以及支原体转化。产生可诱导荧光报告基因的质粒将用于对活力进行评分。