NEW TECHNOLOGIES FOR TIME-RESOLVED INVESTIGATIONS

用于时间分辨调查的新技术

• 批准号: 8172286
• 负责人: TERENCE C WAGENKNECHT
• 金额: $ 37.31万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-07 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8172286
• 关键词: Achievement; Actins; Address; Adsorption; Air; Bacteriorhodopsins; Binding; Biochemical; Biological; Biological Assay; Butterflies; Caliber; Carbon; Characteristics; Cholinergic Receptors; Collaborations; Collection; Computer Retrieval of Information on Scientific Projects Database; Computers; Consumption; Cryoelectron Microscopy; Data Collection; Deposition; Development; Device Designs; Devices; Dimensions; Disease; Electrons; Elements; Environment; Enzymes; Escherichia coli; Evaluation; Ferritin; Figs - dietary; Film; Fluorescein; Fluoresceins; Fluorescence Microscopy; Freezing; Funding; Gases; Generations; Glass; Goals; Grant; Humidity; Ice; Image; Incubated; Institutes; Institution; Investigation; Ions; Label; Laboratories; Left; Liquid substance; Macromolecular Complexes; Malate Dehydrogenase; Methods; Microfluidic Microchips; Microfluidics; Microscopy; Motor; Muscle; Myosin S-1; Nature; Nebulizer; Nitrogen; Partner in relationship; Performance; Physiologic pulse; Plastics; Polymethyl Methacrylate; Power stroke; Process; Protons; Publications; Pump; Reaction; Reaction Time; Reporting; Research; Research Personnel; Resolution; Resources; Ribosomal Proteins; Ribosomes; Sampling; Shapes; Silicon; Simulate; Solutions; Source; Specimen; Spectrum Analysis; Structure; Sucrose; Surface; Syringes; System; Techniques; Technology; Temperature; Testing; Time; Tracer; Ultracentrifugation; United States National Institutes of Health; Walkers; Work; abstracting; aqueous; base; biocompatible polymer; caged molecule; design; experience; hydrophilicity; improved; indexing; macromolecule; millisecond; miniaturize; nanodevice; new technology; novel; operation; particle; premature; pressure; protein function; reaction rate; reconstruction; research study; residence; simulation; small molecule; stopped-flow fluorescence; translation assay; ultraviolet irradiation

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. ABSTRACT: Currently, microfluidics technology is being applied to develop micromixers for time resolved cryo-electron microscopy (TRCEM) application. TRCEM requires fast and homogeneous premixing of tiny amounts of macromolecules in the time scale of milliseconds or less. Work is also just beginning in which this technology will be applied to the problems of specimen deposition onto EM grids and rapid freezing. Our goal for the remainder of this grant is to design and test a microfluidics-based device that allows TRCEM experiments to be conducted routinely with millisecond time resolution and requires only the small amounts of biomolecules that are often available to researchers. The nano-device design and fabrication is conducted in collaboration with Dr. Toh Ming and Dr. Zonghuan Lu at Rensselaer Polytechnic Institute via subcontract from the P41 grant. Very few publications (Tittor et al., 2002; Walker et al., 1999) have appeared over the past decade employing TRCEM on sub-second time scales. Aside from the ongoing efforts in our laboratory, the other existing technologies involve depositing the macromolecular complex on the specimen grid by the standard blotting technique and then initiating the reaction as the grid is being plunged into cryogen by spraying it with a small-molecule reactant (Berriman and Unwin, 1994; White et al., 2003; White et al., 1998) or exposing the grid to a brief pulse of UV irradiation to activate a photo-sensitive reactant, either a chemically synthesized "caged" molecule or a naturally photosensitive component (Menetret et al., 1991; Subramaniam et al., 1993). Probably the most notable achievement to date of sub-second TRCEM was the trapping and structural determination of the acetylcholine receptor in its transiently open state by Unwin over 15 years ago (Unwin, 1995). In large part, the objective of this TRD in the last few years has focused on developing a device that combines rapid mixing and spraying to deposit a thin layer of aqueous biological samples on cryo-EM grids, i.e., to achieve sample mixing in the submillisecond time scale, to incubate the mixture for a particular time scale of milliseconds or more, and then to spray the sample onto TEM grids. A major advantage of the method we have developed is that it allows the reaction to occur in bulk solution and not on the EM grid where reactants can undergo interactions (e.g. irreversible adsorption) to the carbon support film. Fig. 1. Micromixer development. (a) Configuration of the micromixer, with detailed dimensions labeled. SEM images on the bottom show the mixing channel as fabricated using DRIE etching technology (left), and the details of half of the butterfly mixing unit (right). (b) Fluorescence microscopy images of mixing fluorescein and Fluoro Sphere Red in the fabricated mixer at various flowrates. (C) Experimental and simulated mixing indices showing essentially complete mixing at flow rates above 4 ¿L/sec. (D) Activity of malate dehydrogenase is preserved at various flowrates. To achieve this objective, we developed and evaluated numerous microfluidic mixer designs for rapid sample mixing with economical sample volumes. These designs included T- or Tesla-shaped mixers, hemicircular- and butterfly-shaped in-channel mixing elements, and their combinations. Initial evaluations of the designs were done using three-dimensional Computational Fluid Dynamics (CFD) simulations. Guided by the CFD results, physical devices were fabricated using micro-machining technologies (including DRIE, deep reactive ion etching) and were tested to assess their efficiency for rapid mixing. A novel micromixer that combined two T-shaped premixers and four in-channel butterfly mixing elements (Fig. 1a) displayed mixing performance characteristics that should satisfy most requirements of TRCEM. The mixer utilizes a chaotic advection mixing strategy to achieve submillisecond mixing, while sample consumption is small at several Fig. 2. Monolithic microfluidic devices for mixing and spraying (a) Detailed schematic of monolithic device (micromixer/sprayer) design showing (1) device design layout, (2) micromixer configuration, (3) SEM image of the mixer, (4) external atomization nozzle design, (5) SEM image of the nozzle, (6) device top view, and (7) device bottom view. (b) The droplet size distributions for two of the devices with the external atomization nozzles, showing the droplet size mainly in the range of 10-30 ¿m. Fig. 3. Experimental setup with microspray generation shown: (a) Diagram of the experimental system, including syringe pump for liquid transport, monolithic device for mixing and spraying, and cryo-EM grid for droplet collection. (b) Photo of the entire experimental system setup. (c) Close-up of the device with fittings, which is mounted on a plastic holder. A tweezers (connected to the plunger) with an EM grid placed at the exit of the spray nozzle. (d) Fine microspray (blue arrows) generated by the device with the liquid flow rate at 6.0 ¿L/s, and with nitrogen gas pressure at 50 psi (set by nitrogen tank regulator). microliters per second. The simulation results showed that the mixing structures generated chaotic advection in the mixer by forming secondary vortex flows in the microchannels. For example, at a flow rate of 6.0 ¿L/s the calculated mixing index is 99%, and the mean residence time is less than 0.5 msec for 95% of the molecules. Experimental mixing tests of the actual devices indicated that the mixer performance is consistent with the simulation results, as shown in Figure 1 (b,c). To determine whether the devices have deleterious effects on protein function we also conducted enzymatic activity tests of malate dehydrogenase, which was passed through the micromixer at various flow rates up to 10 ¿L/s. Full activity, assessed by comparison to enzyme that had not gone through the mixer, was retained at all flow rates tested (Fig. 1d). These results are a good indicator that the device will be useful for biophysical characterization of the dynamics of functioning or assembling macromolecular complexes. More recent studies using ribosomal protein translation assays and assembly of ribosomal subunits have thus far given no indications of any damage caused by the microdevices. The next challenge was to deposit reaction mixtures that had passed through the micromixers on to EM grids such that they could be frozen with minimal delay. Several years ago our lab developed a mechanically machined air-assisted sprayer consisting of a 100-¿m diameter plastic-tubing liquid nozzle surrounded by a ~300 ¿m conical plastic air nozzle (Barnard and Wagenknecht, 2005). We attempted to mate this sprayer to various mixers (a simple T mixer and one of the micromixers described above), but results were unsatisfactory. For example, this system was impractical for reaction times on the order of milliseconds. However, our experience with this sprayer was valuable. It has led us to the design and fabrication of an integrated monolithic device that combines the micromixers described above with a miniaturized air-assisted sprayer that can achieve processing (mixing + reaction) times as short as a few milliseconds. Silicon and Pyrex glass wafers were used as substrates but other materials can also be used, such as PDMS, PMMA, or other suitable biocompatible polymers. The device's performance for generating micron size droplets was tested. Ribosome-ferritin mixing experiments were carried out to verify its mixing efficiency. Several methods were used to evaluate the activities of biological macromolecules after passing them through the device, including 3D cryo-EM reconstruction, UV-spectroscopy, sucrose-gradient ultracentrifugation, and assays of ribosomal activity. Three types of devices were tested based on their nozzle configurations, including external or internal atomization nozzles. All the devices successfully generated microsprays at flow rates of 4-6 ¿L/s. A solution of ferritin was used as tracer when sprayed on TEM grids to detect the droplet size distribution. The two devices with the external atomization spray nozzle configuration, shown in Figure 2a, more stably generated microdroplet sprays than the other types of sprayers, and yielded adequate coverage of the grid by droplets. The droplet sizes are in the range of tens of micrometers (as shown in Figure 2b), which generally fulfilled the droplet size requirement, although the large number of droplets greater than 25 ¿m in diameter is not optimal for good spreading for cryo-EM. Figure 3 shows the experimental setup for TRCEM, including a photograph of the device in operation while generating a microdroplet-containing spray. Two devices are currently being tested, one with a total processing time of 10 msec and the other 42 msec (about 5 msec of this time is for spraying onto the grid and plunging into cryogen). We are currently utilizing this experimental setup for ribosome subunit association experiments with the goal of discovering intermediate states in the reassociation of 30S and 50S ribosomal subunits to form 70S monosomes. We have successfully imaged and determined 3D reconstructions of ribosomes that were passed through the devices (Figure 4). The resolution attained was the same as that obtained for a similar number of particles imaged by conventional (non-time-resolved) cryo-EM. Further, biophysical and biochemical characterization of ribosomes and ribosomal subunits that have passed through the device also indicated no loss of structural or functional activity. Experiments to characterize the reassociation of the E. coli ribosome from 30S and 50S subunits in the monolithic devices have commenced, and the preliminary results show that reassociation is occurring in the monolithic devices. Figure 5 (top panel) shows some selected examples of images that have been identified as re-assembled ribosomes. Already, some atypical images have been observed, but it is premature to identify these as true intermediates in reassociation. Several issues should be addressed to improve the performance of our current experimental system. First, the average droplet size is larger than is optimal for data collection; larger droplets tend not to spread sufficiently thin for optimal contrast. Related to this issue, enhancing the hydrophilicity of the grid surface would promote better surface wetting and droplet spreading. The most important factors that need to be extensively investigated are, first, time resolution control, which will allow a broader range of biological applications and, second, reduced sample consumption, especially for valuable biological samples whose amounts are usually very limited. Other issues related to system improvement include temperature and humidity control to obtain adjustable and reproducible reaction rates, and to facilitate the integration of monolithic device into the environments commonly found in laboratories that conduct cryo-EM experiments. Fig. 4. Cryo-EM of ribosomes and ferritin that were mixed and sprayed by the monlithic device #2. Upper left: low magnification micrograph showing the edge of a droplet that has spread sufficiently thin for image. Right: High magnification view of a hole containing thin ice and showing presence of both ferritin (dark structures) and ribosomes (lighter structures). Lower left: Surface representation of a three-dimensional reconstruction of the ribosome determined from images such as that shown in B. Resolution is 18.9¿. Adapted from Lu et al. J. Struct. Biol.. References: Barnard D and T Wagenknecht. 2005. Pneumatic Micro-Sprayer for Millisecond Time Resolution in Cryo-Electron Microscopy. Microscopy & Microanalysis 11:290-291. Berriman J and N Unwin. 1994. Analysis of transient structures by cryo-microscopy combined with rapid mixing of spray droplets. Ultramicroscopy 56:241-252. Subramaniam, S., M.Gerstein, D.Oesterhelt, and R.Henderson. 1993. Electron Diffraction Analysis of Structural Changes in the Photocycle of Bacteriorhodopsin. EMBO J. 12:1-8. Tittor, J., S.Paula, S.Subramaniam, J.Heberle, R.Henderson, and D.Oesterhelt. 2002. Proton translocation by bacteriorhodopsin in the absence of substantial conformational changes. J. Mol. Biol. 319:555-565. Unwin, N. 1995. Acetylcholine receptor channel imaged in the open state. Nature 373:37-43. Walker, M., X.-Z.Zhang, W.Jiang, J.Trinick, and H.D.White. 1999. Observation of transient disorder during myosin subfragment-1 binding to actin by stopped-flow fluorescence and millisecond time resolution cryomicroscopy: evidence that the start of the crossbridge power stroke in muscle has variable geometry. Proc. Natl. Acad. Sci. USA 96:465-470. White, H.D., K.Thirumurugan, M.L.Walker, and J.Trinick. 2003. A second generation apparatus for time-resolved electron cryo-microscopy using stepper motors and electrospray. J. Struct. Biol. 144:246-252. White, H.D., M.L.Walker, and J.Trinick. 1998. A computer-controlled spraying-freezing apparatus for millisecond time-resolution electron cryomicroscopy. J. Struct. Biol. 121:306-313. In the previous reporting period, the following abstracts were presented: + Lu, Z., McMahon, J., Mohamed, H., Barnard, D., Shaikh, T.R., Wagenknecht, T. and Lu, T.M. 2008. Microfluidic Mixing System for Time Resolved Cryo-Electron Microscopy. Microscopy and Microanalysis 14:1598-1599. + Barnard, D., Lu, Z., Shaikh, T.R., Mohamed, H., Buttle, K., McMahon, J., Meng, X., Lu, T.M. and Wagenknecht, T. 2008. 1576-Pos Development of a Reaction Mixer/Micro-Nebulizer for Time-Resolved Cryo-Electron Microscopy of Macromolecular Systems. Biophys. J. 94:1576.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以出现在其他 CRISP 条目中 列出的机构是。 对于中心来说，它不一定是研究者的机构。 抽象的： 目前，微流体技术正在应用于开发用于时间分辨冷冻电子显微镜（TRCEM）应用的微混合器，该应用需要在毫秒或更短的时间尺度内快速均匀地预混合微量大分子。技术将应用于解决样品沉积到电磁网格上和快速冷冻的问题，我们的剩余资金目标是设计和测试基于微流体的设备，该设备允许。 TRCEM 实验通常以毫秒时间分辨率进行，只需要研究人员通常可以获得的少量生物分子。纳米器件的设计和制造是与伦斯勒理工学院的 Toh Ming 博士和 Zonghuan Lu 博士合作进行的。通过 P41 拨款的分包。 在过去的十年中，很少有出版物（Tittor 等人，2002；Walker 等人，1999）在亚秒级时间尺度上使用 TRCEM，除了我们实验室正在进行的努力之外，其他现有技术涉及沉积大分子。通过标准印迹技术在样品网格上形成复合物，然后在将网格浸入冷冻剂时通过喷洒小分子反应物来启动反应（Berriman 和 Unwin， 1994；White et al.，2003；White et al.，1998）或将网格暴露于短暂的紫外线照射下以激活光敏反应物，可以是化学合成的“笼状”分子或天然光敏成分（Menetret）亚秒 TRCEM 迄今为止最显着的成就可能是捕获和Unwin 于 15 年前对短暂开放状态的乙酰胆碱受体进行了结构测定（Unwin，1995）。 在很大程度上，过去几年该TRD的目标集中在开发一种将快速混合和喷雾相结合的装置，以在冷冻电镜网格上沉积一层薄薄​​的水性生物样品，即在亚毫秒内实现样品混合我们开发的方法的一个主要优点是，它允许反应在本体溶液中发生，并且可以在特定的时间范围内进行。不在电磁网格上，反应物可以与碳支撑膜发生相互作用（例如不可逆吸附）。 图 1. 微混合器的开发。 (a) 微混合器的配置，底部标有详细尺寸的 SEM 图像显示使用 DRIE 蚀刻技术制造的混合通道（左），以及一半蝶形混合单元的细节（ (b) 在不同流速下在制造的混合器中混合荧光素和荧光球红的荧光显微镜图像 (C) 实验和模拟混合指数显示在流动时基本完全混合。利率高于 4 ¿ L/sec. (D) 苹果酸脱氢酶的活性在不同的流速下得以保留。 为了实现这一目标，我们开发并评估了多种微流体混合器设计，用于以经济的样品体积快速混合样品，这些设计包括 T 形或特斯拉形混合器、半圆形和蝴蝶形通道内混合元件及其组合。使用三维计算流体动力学 (CFD) 模拟对设计进行评估，在 CFD 结果的指导下，使用微加工技术（包括 DRIE、深反应）制造物理设备。离子蚀刻）并进行了测试，以评估其快速混合的效率。一种新型微混合器结合了两个 T 形预混合器和四个通道内蝶形混合元件（图 1a），其混合性能特征应满足 TRCEM 混合器的大多数要求。采用混沌平流混合策略实现亚毫秒级混合，同时样品消耗量较小 图 2. 用于混合和喷雾的整体式微流体装置 (a) 整体式装置（微混合器/喷雾器）设计的详细示意图，显示 (1) 装置设计布局，(2) 微混合器配置，(3) 混合器的 SEM 图像，(4) ) 外部雾化喷嘴设计，(5) 喷嘴的 SEM 图像，(6) 器件俯视图，(7) 器件仰视图，(b) 两种液滴尺寸分布。具有外部雾化喷嘴的装置，显示液滴尺寸主要在10-30 ¿米。 图 3. 显示微喷雾生成的实验装置：(a) 实验系统图，包括用于液体输送的注射泵、用于混合和喷雾的整体装置以及用于液滴收集的冷冻电镜网格。 (c) 带有配件的装置的特写，该装置安装在塑料支架上，镊子（连接到柱塞）在喷雾出口处放置有电磁网格。 (d) 装置产生的精细微喷雾（蓝色箭头），液体流速为 6.0 ¿ L/s，氮气压力为 50 psi（由氮气罐调节器设置）。 模拟结果表明，混合结构通过在微通道中形成二次涡流而在混合器中产生混沌平流。例如，流速为 6.0 ¿ L/s计算的混合指数为99%，95%的分子的平均停留时间小于0.5毫秒。实际装置的实验混合测试表明，混合器的性能与模拟结果一致，如图所示。图 1 (b,c)。 为了确定这些设备是否对蛋白质功能产生有害影响，我们还进行了苹果酸脱氢酶的酶活性测试，该酶以高达 10 ¿ 的各种流速通过微混合器。 L/s。通过与未经过混合器的酶进行比较来评估，在所有测试的流速下都保留了完整的活性（图 1d），这些结果是一个很好的指标，表明该设备将可用于生物物理表征。迄今为止，使用核糖体蛋白质翻译测定和核糖体亚基组装的最新研究尚未表明微型装置会造成任何损害。 下一个挑战是将通过微混合器的反应混合物沉积到 EM 网格上，以便可以以最小的延迟进行冷冻。几年前，我们的实验室开发了一种由 100-¿ 组成的机械加工空气辅助喷雾器。米直径的塑料管液体喷嘴，周围环绕着约 300 ¿ m 锥形塑料空气喷嘴（Barnard 和 Wagenknecht，2005）我们尝试将这种喷雾器与各种混合器（简单的 T 型混合器和上述微型混合器之一）配合使用，但结果并不令人满意，例如，该系统对于反应来说是不切实际的。然而，我们在这款喷雾器方面的经验非常宝贵，它引导我们设计和制造了一种集成的整体设备。上述微混合器带有微型空气辅助喷雾器，可实现短至几毫秒的处理（混合+反应）时间，使用硅和 Pyrex 玻璃晶片作为基材，但也可以使用其他材料，例如 PDMS、PMMA、测试了该装置产生微米尺寸液滴的性能，以验证其混合效率。大分子通过设备后进行分析，包括 3D 冷冻电镜重建、紫外光谱、蔗糖梯度超速离心和核糖体活性测定。 根据喷嘴配置（包括外部或内部雾化喷嘴）对三种类型的设备进行了测试，所有设备均成功产生了 4-6 ¿ L/s。当喷射到 TEM 网格上时，使用铁蛋白溶液作为示踪剂来检测液滴尺寸分布，如图 2a 所示，两种具有外部雾化喷嘴配置的装置比其他类型的液滴喷雾更稳定。喷雾器，并产生足够的液滴尺寸覆盖网格（如图2b所示），这通常满足液滴尺寸要求。大量液滴大于 25 ¿ m 的直径对于冷冻电镜的良好扩散来说并不是最佳的。 图 3 显示了 TRCEM 的实验设置，包括正在运行的设备的照片，同时生成含有微滴的喷雾。目前正在测试两个设备，一个的总处理时间为 10 毫秒，另一个的总处理时间为 42 毫秒（约 5 毫秒）。这次用于喷射到网格上并投入冷冻剂）我们目前正在利用该实验装置进行核糖体亚基关联实验，目的是发现核糖体重新关联中的中间状态。 30S和50S核糖体亚基形成70S单体。 我们成功地对通过设备的核糖体进行了成像并确定了 3D 重建（图 4），所获得的分辨率与传统（非时间分辨）冷冻电镜成像的类似数量的颗粒所获得的分辨率相同。此外，通过该装置的核糖体和核糖体亚基的生物物理和生化特征也表明没有损失结构或功能活性。 表征单片器件中 30S 和 50S 亚基的大肠杆菌核糖体重关联的实验已经开始，初步结果表明重关联正在单片器件中发生。图 5（上图）显示了一些选定的图像示例。已经被鉴定为重新组装的核糖体，已经观察到一些非典型图像，但将它们鉴定为重新结合的真正中间体还为时过早。 为了提高我们当前实验系统的性能，需要解决几个问题：平均液滴尺寸大于数据收集的最佳尺寸；与此问题相关的是，较大的液滴往往无法铺展得足够薄，从而增强了亲水性。网格表面的优化将促进更好的表面润湿和液滴扩散，首先是时间分辨率控制，这将允许更广泛的生物应用，其次是减少样品消耗，特别是对于有价值的样品。生物样本的数量通常非常有限。与系统改进相关的其他问题包括温度和湿度控制，以获得可调节和可重复的反应速率，并促进将单片设备集成到进行冷冻电镜实验的实验室中常见的环境中。 图 4. 通过整体式装置 #2 混合和喷射的核糖体和铁蛋白的冷冻电镜图。 左上：低放大倍率显微照片，显示已铺展得足够薄的液滴边缘，右图：高放大倍率视图的图像。含有薄冰的孔，显示铁蛋白（深色结构）和核糖体（浅色结构）的存在。左下：根据图像确定的核糖体三维重建的表面表示。如B所示。分辨率为18.9¿改编自 Lu 等人，《结构》。 参考： Barnard D 和 T Wagenknecht。2005 年。冷冻电子显微镜中毫秒时间分辨率的气动微型喷雾器 11：290-291。 Berriman J 和 N Unwin。1994 年。通过冷冻显微镜与快速混合喷雾液滴分析瞬态结构。56：241-252。 Subramaniam, S.、M.Gerstein、D.Oesterhelt 和 R.Henderson，1993 年。细菌视紫红质光循环结构变化的电子衍射分析。12:1-8。 Tittor, J.、S. Paula、S. Subramaniam、J. Heberle、R. Henderson 和 D. Oesterhelt。2002 年，细菌视紫红质在没有显着构象变化的情况下发生质子易位。 565. Unwin, N. 1995。乙酰胆碱受体通道在开放状态下成像，Nature 373:37-43。 Walker, M., X.-Z. 张、W. Jiang、J. Trinick 和 H. D. White 1999。通过停流荧光和毫秒时间分辨率冷冻显微镜观察肌球蛋白亚片段 1 与肌动蛋白结合过程中的短暂紊乱：证据肌肉中的横桥动力冲程的开始具有可变的几何形状。 White, H.D.、K. Thirumurugan、M.L. Walker 和 J. Trinick。使用步进电机和电喷雾的第二代设备。J. Biol。 White，H.D.，M.L.Walker，和 J.Trinick。用于毫秒时间分辨率电子冷冻显微镜的计算机控制喷雾冷冻装置。J.Struct。 上一报告期，提交的摘要如下： + Lu, Z.、McMahon, J.、Mohamed, H.、Barnard, D.、Shaikh, T.R.、Wagenknecht, T. 和 Lu, T.M. 2008。用于时间分辨冷冻电子显微镜和微量分析的微流体混合系统 14。 ：1598-1599。 + Barnard, D.、Lu, Z.、Shaikh, T.R.、Mohamed, H.、Buttle, K.、McMahon, J.、Meng, X.、Lu, T.M. 和 Wagenknecht, T. 2008。1576-Pos 开发用于大分子系统时间分辨冷冻电子显微镜的反应混合器/微雾化器。 94:1576。