Laser-free Ultrafast Tunable Stroboscopic TEM imaging for Biomedical Applications

适用于生物医学应用的无激光超快可调谐频闪 TEM 成像

• 批准号: 10242205
• 负责人: Chunguang Jing
• 金额: $ 76.48万
• 依托单位: EUCLID BEAMLABS, LLC
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-01 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10242205
• 关键词: Address; Apoferritin; Award; Biocompatible Materials; Biological; Biology; Biomaterials Research; Biomedical Research; Biomedical Technology; California; Cellular biology; Communities; Computing Methodologies; Cryoelectron Microscopy; Crystallization; Dependence; Development; Dose; Dose-Rate; Electron Beam; Electron Microscope; Electron Microscopy; Electrons; Engineering; Face; Freezing; Future; Generations; Grant; Health; Hydration status; Image; Institutes; Investigation; Laboratories; Lasers; Legal patent; Link; Methods; Microscopy; Modernization; Modification; Molecular; Movement; Noise; Paraffin; Phase; Physics; Physiologic pulse; Process; Purple Membrane; Radiation induced damage; Research; Research Personnel; Resolution; Sampling; Series; Signal Transduction; Small Business Innovation Research Grant; Specimen; Streptavidin; Structure; System; Techniques; Technology; Temperature; Testing; Three-Dimensional Imaging; Time; Training; Transmission Electron Microscopy; United States National Institutes of Health; University Hospitals; Water; Width; base; beta-Galactosidase; bioimaging; biological research; biomacromolecule; cellular imaging; commercialization; cost effective; cryogenics; crystallinity; density; design; experience; imaging modality; improved; innovation; insight; irradiation; microscopic imaging; movie; nanoscale; next generation; novel; particle; prevent; research and development; success; tomography; tool; transmission process; vibration

Title: Laser-free Ultrafast Tunable Stroboscopic TEM imaging for Biomedical Applications PI: C. Jing Project Summary/Abstract Major advances in cell biology and biomedical research are tightly linked to innovations in microscopy. Modern cryogenic transmission electron microscopy (cryo-EM) achieves near-atomic-resolution images but faces key barriers. Beam-induced radiation damage during image exposure limits higher resolution: useful signal added per incident electron decreases due to damage, while added noise remains roughly constant, meaning an optimum exposure exists beyond which signal-to-noise worsens. Cryotomographic techniques allow 3D imaging, but their tilted images can suffer from vibrational blur. And ultrafast transmission electron microscopy (UTEM) now takes “molecular movies” rather than static images by using femtosecond lasers, but with the exorbitant expense and invasive modifications; only a handful exist worldwide and image acquisition is slow due to typical 1MHz or lower laser repetition rates. Euclid Beamlabs, LLC, is addressing all these challenges by developing a single technology: a laser-free, cost-effective, retrofittable TEM pulser, widely tunable from Herz to Gigahertz repetition rate and microsecond to picosecond pulse duration. Our first-generation pulser won a 2019 R&D 100 Award. The NIH SBIR Phase I project from July to November 2019 demonstrated first bio-imaging on two Euclid-retrofitted JEOL TEMs. We showed irradiation damage mitigation of C36H74 paraffin and purple membrane: compared to continuous beam, our GHz pulsed beam produced up to 2.5x less irradiation damage at equal dose, repeatably tested to 10 electrons per square Angstrom (10e-/Å2) at both 200 and 300 kV. Phase II will build on the successes of Phase I. We will introduce second-generation pulser technology in a cryo-TEM for the first time. The dynamic range of pulse duty factor will be ten orders of magnitude higher than in Phase I using our newly patented Pulse Picker, an essential requirement to address the previously introduced cryo-EM imaging limitations. We will produce a pulsed beam suitable for vibration-insensitive cryotomography, potentially allowing sharper images and full tilt-series in seconds. We will optimize pulse structure for reduced radiation damage and higher critical dose, leading to higher contrast and higher resolution. We will assess whether a pulsed beam also improves vitreous water crystallization, a canonical cryo-EM limitation. And we will quantify temperature- and pulse-dependent radiation damage interrelationships for crystalline and single-particle bio-samples. At the conclusion of Phase II, the new pulsed cryo-EM and the many proof-of-principle use cases will initiate commercialization of this affordable, retrofittable, versatile system to bring high resolution, high contrast, vibration-insensitive, and time-resolved electron microscopy to the wider bio- imaging community.

标题：用于生物医学的无激光超快可调谐频闪 TEM 成像 应用领域 PI：C. Jing 项目概要/摘要 细胞生物学和生物医学研究的重大进展与现代显微镜的创新密切相关。 低温透射电子显微镜（cryo-EM）可实现近原子分辨率的图像，但面临关键障碍。 图像曝光期间光束引起的辐射损伤限制了更高的分辨率：每次事件添加有用信号 电子因损坏而减少，而添加的噪声大致保持不变，这意味着存在最佳曝光 超过此值，冷冻断层扫描技术允许 3D 成像，但其倾斜图像可能会受到影响。 超快透射电子显微镜 (UTEM) 现在采用的是“分子电影”。 与使用飞秒激光的静态图像相比，但费用高昂且需要进行侵入性修改； 全球范围内存在少数这种激光器，但由于典型的 1MHz 或更低的激光重复率，图像采集速度很慢。 Euclid Beamlabs, LLC 正在通过开发一种单一技术来应对所有这些挑战：一种无激光、经济高效、 可改装 TEM 脉冲发生器，可从赫兹到千兆赫重复率和微秒到皮秒脉冲进行广泛调谐 我们的第一代脉冲发生器于 7 月至 11 月荣获 2019 年 R&D 100 奖。 2019 年，在两台经过 Euclid 改造的 JEOL TEM 上首次展示了生物成像，我们展示了辐射损伤的减轻。 C36H74 石蜡和紫色膜：与连续光束相比，我们的 GHz 脉冲光束产生的能量最多减少 2.5 倍 等剂量辐照损伤，在 200 和 300 kV 下以每平方埃 10 个电子 (10e-/Å2) 进行重复测试。 第二阶段将建立在第一阶段成功的基础上。我们将在冷冻 TEM 中引入第二代脉冲发生器技术 脉冲占空因数的动态范围将首次比第一阶段高出十个数量级。 新获得专利的脉冲选择器是解决之前引入的冷冻电镜成像限制的基本要求。 我们将生产适用于振动不敏感冷冻断层扫描的脉冲束，有可能获得更清晰的图像 我们将在几秒钟内优化脉冲结构，以减少辐射损伤并提高临界剂量， 我们将评估脉冲光束是否也能改善玻璃水。 结晶，这是一种典型的冷冻电镜限制，我们将量化与温度和脉冲相关的辐射损伤。 在第二阶段结束时，新的脉冲冷冻电镜研究了晶体和单颗粒生物样品的相互关系。 许多原理验证用例将启动这种经济实惠、可改造、多功能系统的商业化 将高分辨率、高对比度、振动不敏感和时间分辨电子显微镜应用到更广泛的生物领域 影像社区。