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: 10082035
• 负责人: Chunguang Jing
• 金额: $ 77.99万
• 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 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10082035
• 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频闪透射电子显微镜成像 应用 派：C.Jing 项目摘要/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)获得了接近原子分辨率的图像，但面临着关键的障碍。 图像曝光过程中的束致辐射损伤限制了更高的分辨率：每次入射添加有用信号 电子因损伤而减少，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，信噪比就会恶化。冷冻断层摄影技术可以进行3D成像，但倾斜的图像可能会受到影响 来自振动模糊。而超快透射电子显微镜(UTEM)现在拍摄的是“分子电影” 比使用飞秒激光的静态图像更好，但具有昂贵的费用和侵入性的修改；只有 由于典型的1 MHz或更低的激光重复频率，全球范围内存在少数几个这样的图像，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：无激光、成本效益高、 可改装的瞬变电磁脉冲发生器，可从赫兹到千兆赫兹重复频率和微秒到皮秒脉冲广泛可调 持续时间。我们的第一代脉冲器获得了2019年研发100奖。7-11月NIH SBIR第一阶段项目 2019年在两个欧几里德改装的JEOL TEM上展示了第一次生物成像。我们展示了辐射损害缓解 C36H74石蜡和紫膜：与连续光束相比，我们的GHz脉冲光束的产生量最多减少2.5倍 同等剂量的辐射损伤，在200千伏和300千伏时，重复测试为每平方埃(10e-/a2)10个电子。 第二阶段将建立在第一阶段的成功基础上。我们将在低温瞬变电子显微镜中引入第二代脉冲发生器技术 这是第一次。脉冲占空比的动态范围将比第一阶段高出十个数量级 最新获得专利的Pulse Picker，这是解决之前引入的低温电磁成像限制的基本要求。 我们将生产一种适用于振动不敏感的低温层析成像的脉冲光束，潜在地允许更清晰的图像 和全倾斜系列在几秒钟内。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，一个典型的低温电磁限制。我们将量化依赖于温度和脉冲的辐射损害 晶体和单颗粒生物样品的相互关系。在第二阶段结束时，新的脉冲低温电磁 许多原则证明用例将启动这个负担得起的、可改装的、多功能的系统的商业化 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 影像社区。