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 PI：C。景 项目总结/摘要 细胞生物学和生物医学研究的重大进展与显微镜的创新密切相关。现代 低温透射电子显微镜（cryo-EM）实现了近原子分辨率的图像，但面临着关键的障碍。 图像曝光过程中的光束诱导辐射损伤限制了更高的分辨率：每次事件都增加了有用的信号 电子由于损坏而减少，而增加的噪声大致保持不变，这意味着存在最佳曝光 超过这个范围，信噪比就会下降。冷冻层析成像技术允许3D成像，但其倾斜的图像可能会受到影响 振动模糊超快透射电子显微镜（UTEM）现在可以拍摄“分子电影”， 比静态图像使用飞秒激光，但与过高的费用和侵入性修改;只有一个 世界范围内存在少数，且由于典型的1 MHz或更低的激光重复率，图像获取缓慢。 欧几里得Beamlabs有限责任公司正在通过开发一种单一的技术来解决所有这些挑战：一种无激光、具有成本效益、 可改装的TEM脉冲发生器，重复频率可从赫兹到千兆赫，脉冲可从微秒到皮秒进行广泛调谐 持续时间我们的第一代脉冲发生器荣获2019年R&D 100奖。NIH SBIR第一阶段项目，7月至11月 2019年，在两台Euclid-restored JEOL TEM上首次展示了生物成像。我们发现， C36 H74石蜡和紫膜：与连续光束相比，我们的GHz脉冲光束产生的能量减少了2.5倍 在相同剂量下的辐照损伤，在200和300 kV下重复测试至每平方埃10个电子（10 e-/Ω 2）。 第二阶段将以第一阶段的成功为基础。我们将在低温TEM中引入第二代脉冲发生器技术 第一次脉冲占空因数的动态范围将比使用我们的 新获得专利的Pulse Picker是解决先前引入的冷冻EM成像限制的基本要求。 我们将生产一种脉冲光束，适用于振动不敏感的冷冻断层扫描，可能使图像更清晰 和完整的倾斜系列。我们将优化脉冲结构以减少辐射损伤和提高临界剂量， 导致更高的对比度和更高的分辨率。我们将评估脉冲光束是否也能改善玻璃体水 结晶，一个典型的cryo-EM限制。我们将量化温度和脉冲相关的辐射损伤 晶体和单颗粒生物样品的相互关系。在第二阶段结束时，新的脉冲低温EM 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的电子显微镜带到更广泛的生物学领域， 影像社区