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

项目摘要 该提案描述了一项计划，以开发一种高分辨率可视化活细胞生理学的方法 使用集成的微分相位对比扫描透射电子显微镜（iDPC-STEM）在低 剂量，以促进生存能力。可视化生理学需要具有相称深度的空间分辨率， 场的规模的蛋白质机器（3-7纳米），驱动它没有伴随的损害。与 将包含水的液体流动池引入由膜制成的真空密封外壳中，所述膜 透明的电子束，应该有可能仔细检查生物与高分辨率下， 生理条件与STEM。该提案侧重于三个具体的技术挑战，测试 在充分表征的生物系统的坩埚中的溶液： 1.使用由超薄膜和薄垫片形成的液体流动池提高分辨率。到 减少膜和液体中的散射，使氮化硅（SiN）膜收缩是可行的 形成8-10 nm的液体单元，并且将它们间隔150 nm而不损害窗口完整性。到 消除在装有流体的液体单元中的膨胀，窗口将用厚肋加强， 可跨越面积大于400平方米。然而，即使是10 nm的SiN膜对于高分辨率来说仍然太厚 显像因此，（3 nm）薄非晶硅（a-Si）和原子级薄石墨烯或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 泰坦干通过可视化三磷酸腺苷（ATP）和荧光链霉亲和素（STR）。 2.通过iDPC-STEM成像提高对比度。为了增加透明生物样品的可见度， 将采用用于成像的相位对比方法iDPC-STEM，该方法使用四象限（分段）分割， 检测器，用于测量相位对象的梯度。iDPC-STEM拥有更高的信噪比， 传统的STEM，这提供了极低剂量成像的可能性。分辨率、对比度和 将在畸变校正的、配备iDPC的Themis Z（60 pm）中测试伴随损伤 通过在薄（0-50 nm厚）液体层中可视化ATP和荧光STR来测量分辨率）。 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 原核细胞如果电子探针与液体池中顶部膜处的细胞相互作用，则高- 可以以这种方式捕获分辨率图像。由于探头沿光轴沿着很浅， 系列也可用于对细胞进行切片以进行3D断层摄影。为了验证这些想法， (100将在浅（150 nm）流动池中培养，并用iDPC-STEM可视化，以发现 它们的纳米结构在感染中发挥的作用。在这种厚的标本中，可能需要多切片模拟， 通知结构。暴露于光束后，进行LIVE/DEAD试验，沿着支原体转化 产生诱导型荧光报告基因的质粒将用于对活力进行评分。

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.