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

项目摘要 该提案描述了一种开发高分辨率显示活细胞生理学的方法的计划 集成差示相差扫描电子显微镜(IDPC-STEM)在低电压下的应用 提高生存能力的剂量。可视化生理学需要空间分辨率和与之相称的深度 在蛋白质机械(3-7纳米)的尺度上驱动它而不伴随损害的场。与 介绍了一种液体流动池，该池包含水在真空密封的封套中，所述封套由 对于电子束来说是透明的，应该可以在 STEM的生理状况。该提案侧重于三个具体的技术挑战，即测试 在具有良好特性的生物系统的熔炉中的解决方案： 1.使用由超薄薄膜和薄间隔物形成的液体流动池来提高分辨率。至 为了减少膜和液体中的散射，缩小氮化硅(SiN)膜是可行的 将液体电池形成8-10 nm，并在不影响窗口完整性的情况下将它们间隔150 nm。至 消除在装满液体的液体槽中鼓包，窗户将用厚厚的肋骨加固，使大 &gt；400平方米的面积可以跨越。然而，即使是10纳米的氮化硅薄膜仍然太厚，无法获得高分辨率 成像。因此，(3 Nm)薄的非晶硅(a-Si)和原子薄的石墨烯或h-BN膜跨越 由SIN形成的肋骨将用作高分辨率成像的窗口。该分辨率将使用 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成像对比度。为了提高透明生物样本的能见度， 成像将采用四象限(分段)分裂的相衬方法IDPC-STEM。 测量位相物体倾斜度的探测器。与IDPC-STEM相比，它具有更高的信噪比 与传统的STEM相比，它提供了极低剂量成像的可能性。分辨率、对比度和 伴随的损伤将在像差校正的、配备IDPC的Themis Z(60 PM)中进行测试 通过在薄(0-50 nm厚)液层中可视化ATP和荧光STR，可以获得更高的分辨率。 3.最后，低剂量的IDPC-STEM将与超薄液体流动池一起用于可视化最小的 原核细胞。如果电子探针与液体电池中顶膜上的电池相互作用，则高- 可以通过这种方式捕获分辨率图像。因为探测器沿着光轴是如此浅，一个焦点 系列还可用于对细胞进行切片以进行3D层析成像。为了验证这些想法，四种支原体 (100 nm大小)将在浅(150 Nm)Flow细胞中培养，并用IDPC-STEM进行可视化，以发现 它们的纳米结构在感染中扮演着重要的角色。在样品中，这种厚的多切片模拟可能需要 通知结构的情况。暴露于电子束后，活/死分析与支原体一起转化 产生诱导性荧光报告的质粒将被用来评估生存能力。

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.