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; 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.

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 抽象的： 目前，正在应用微流体技术来开发用于时间分辨率的低温电子显微镜（TRCEM）应用的微弹器。 TRCEM需要在毫秒或更短的时间尺度上快速且均匀的大量大分子进行均匀的预混合。工作也只是开始，将该技术应用于标本沉积问题上的问题和快速冻结。我们对该赠款的其余部分的目标是设计和测试基于微流体的设备，该设备允许TRCEM实验以毫秒的时间分辨率进行定期进行，并且仅需要研究人员通常可以使用的少量生物分子。纳米设备设计和制造是通过P41 Grant的分包合同与Rensselaer Polytechnic Institute的Toh Ming博士和Zonghuan Lu博士合作进行的。 在过去的十年中，很少有出版物（Tittor等，2002; Walker等，1999）出现在次秒时量表上。除了我们实验室的持续努力外，其他现有技术还涉及通过标准印迹技术将大分子复合物沉积在标本网格上，然后随着网格的污染作用，随着网格被浸入低温而引发反应，通过用小分子反应剂喷雾（Berriman and 1994; White et al。为了激活光敏反应物，是化学合成的“笼”分子或天然光敏的成分（Menetret等，1991； Subramaniam等，1993）。迄今为止，第秒trcem迄今为止最值得注意的成就是15年前UNWIN在其瞬时开放状态下对乙酰胆碱受体的捕获和结构确定（Unwin，1995）。 在很大程度上，在过去几年中，该TRD的目的集中在开发一种结合快速混合和喷涂的设备上，以将一层薄层的水性生物学样品沉积在冷冻EM网格上，即，即以亚毫秒的时间尺度实现样品混合，以将混合物的混合物添加到特定的时间尺度上，以便将毫秒的特定时间缩放到毫秒中，然后将其喷涂到更高的样品中。我们开发的方法的一个主要优点是，它允许反应发生在大量溶液中，而不是在EM网格中发生，在该反应物可以在碳支持膜中进行相互作用（例如不可逆的增加吸附）。 图1。微质子的开发。 （a）Micromixer的配置，标有详细的尺寸。底部的SEM图像显示了使用DRIE蚀刻技术（左）制造的混合通道以及蝴蝶混合单元的一半的细节（右）。 （b）在各种流量处，在制成的混合器中混合荧光素和荧光球的荧光显微镜图像。 （c）实验和模拟混合指数，显示以高于4/sec的流速基本完全混合。 （d）在各种流量范围内保留苹果酸脱氢酶的活性。 为了实现这一目标，我们开发并评估了许多微流体混合器设计，以快速样品与经济样品体积混合。这些设计包括T-或特斯拉形的搅拌机，半圆形和蝴蝶形的渠道内混合元件及其组合。使用三维计算流体动力学（CFD）模拟进行了设计的初步评估。在CFD结果的指导下，使用微型机械手术技术（包括DRIE，深层反应离子蚀刻）制造了物理设备，并经过测试以评估其快速混合的效率。一种新型的微弹器，结合了两个T形预混合物和四个渠道内蝴蝶混合元件（图1A），表现出应满足TRCEM大多数要求的混合性能特征。混音器利用混乱的广告混合策略来实现亚略加混合，而样品消费量很小。 图2。用于混合和喷涂的单层微流体设备（A）单片设备（Micromixer/Sprayer）设计的详细示意图，显示（1）设备设计布局，（2）Micromixer配置，（3）混合器的SEM图像，（4）外部雾化器设计，（4）Notught opmiation notization notization notization nomzization nopsization nomzal Design，（5）notuke sem sep of notuke seption seption，（5）设备的设备，（6）设备（6）设备（6）设备，（6）。 （b）带有外部雾化喷嘴的两个设备的液滴尺寸分布，主要显示液滴尺寸在10-30€m的范围内。 图3。显示的实验设置显示：（a）实验系统的图，包括用于液体传输的注射器泵，用于混合和喷涂的单片设备以及用于液滴收集的冷冻EM网格。 （b）整个实验系统设置的照片。 （c）设备的特写配件，该设备安装在塑料支架上。一个镊子（连接到塞子），将EM网格放在喷嘴的出口处。 （d）设备在6.0€l/s的设备产生的细微喷涂（蓝色箭头），并在50 psi（由氮气罐调节器设置）的氮气压力下。 每秒微层。仿真结果表明，混合结构通过在微通道中形成次级涡流流在混合器中产生混乱的广告。例如，以6.0»l/s的流速为99％，计算出的混合指数为99％，对于95％的分子，平均停留时间小于0.5毫秒。实际设备的实验混合测试表明，混合器性能与仿真结果一致，如图1（B，C）所示。 为了确定这些设备是否删除了对蛋白质功能的影响，我们还进行了苹果酸脱氢酶的酶活性测试，该测试以多种流速（最高10»l/s）通过了微米物。在所有流速测试的所有流速下，都保留了与未经混合器的酶进行比较的全面活性（图1D）。这些结果很好地表明，该设备将有助于对大分子或组装大分子配合物的动力学表征。迄今为止，使用核糖体蛋白翻译测定法和核糖体亚基的组装的最新研究尚未迹象表明该微论述造成的任何损害。 下一个挑战是沉积通过微弹器的反应混合物到EM网格，以便可以以最小的延迟来冷冻它们。几年前，我们的实验室开发了一种机械加工的空气辅助喷雾器，该喷雾器由100-`M直径的塑料管液体喷嘴组成，周围是〜300»圆锥形塑料空气喷嘴（Barnard and Wagenknecht，2005）。我们试图将此喷雾器与各种混合器（简单的T混合器和上面描述的一个微型物）配对，但结果不令人满意。例如，该系统对于毫秒级的反应时间是不切实际的。但是，我们在该喷雾器方面的经验很有价值。它导致我们设计和制造了一种集成的整体设备，该设备将上述微型物与微型空气辅助喷雾器结合在一起，该喷雾器可以达到处理（混合 +反应）的短时间，尽可能短几毫秒。硅和Pyrex玻璃振动剂被用作底物，但也可以使用其他材料，例如PDMS，PMMA或其他合适的生物相容性聚合物。测试了该设备用于生成微米大小液滴的性能。进行了核糖体铁蛋白混合实验，以验证其混合使用几种方法，以评估生物大分子通过该设备的生物学大分子的活性，包括3D冷冻 - 光谱重建，UV-光谱，蔗糖糖散发性超脱口酸和核糖体活动的测定。 根据其喷嘴配置（包括外部或内部雾化喷嘴）测试了三种类型的设备。所有设备都以4-6»l/s的流速成功生成了微刺。当在TEM网格上喷涂铁蛋白的溶液用作示踪剂以检测液滴尺寸的分布。带有外部雾化喷嘴配置的两个设备如图2A所示，比其他类型的喷雾器更稳定地产生的微螺旋式喷雾剂，并通过液滴对网格进行了足够的覆盖。液滴尺寸在数十毫米的范围内（如图2B所示），尽管大量的直径大于25€m的大量液滴并不是良好的散布，但对于cryo-em而言，液滴尺寸的需求通常通常满足。 图3显示了TRCEM的实验设置，包括在生成含微螺旋杆的喷雾时运行的设备照片。目前正在测试两种设备，一台总处理时间为10毫秒，其他42毫秒（这次约5毫秒都用于喷涂到网格上并浸入冷冻原）。我们目前正在利用这种实验设置进行核糖体亚基关联实验，目的是在30S和50S核糖体亚基重新关联中发现中间状态以形成70S单子体。 我们已经成功成像并确定了通过设备的核糖体的3D重建（图4）。该分辨率与传统（非时期分辨）冷冻EM成像的类似数量的颗粒获得的分辨率相同。此外，通过设备的核糖体和核糖体亚基的生物物理和生化表征也表明结构或功能活性没有损失。 已经开始表征整体设备中30s和50s亚基的大肠杆菌核糖体的重新关联的实验，并且初步结果表明，在整体设备中发生了重新关联。图5（顶部面板）显示了一些已确定为重新组装核糖体的图像的选定示例。已经观察到了一些非典型图像，但是将其确定为核糖体中的真实中间体已经为时过早。 应该解决几个问题，以提高我们当前的实验系统的性能。首先，平均液滴尺寸大于数据收集最佳。较大的液滴往往不会适当地散布，从而获得最佳对比度。与此问题相关，增强网格表面的亲水性将促进更好的表面润湿和液滴扩散。首先，需要进行广泛研究的最重要因素是时间分辨率控制，这将允许更广泛的生物应用，其次可以减少样品消耗，尤其是对于有价值的生物样品通常非常有限的有价值的生物样品。与系统改进有关的其他问题包括温度和湿度控制，以获得可调节和可再现的反应速率，并促进整体设备的整合到进行冷冻EM实验的实验室中常见的环境中。 图4。核糖体和铁蛋白的冷冻EM被蒙特利思上装置＃2混合并喷洒。左上：低放大显微照片显示液滴的边缘，该液滴散布了足够薄的图像。右：含有薄冰的孔的高放大倍率，并显示铁蛋白（深色结构）和核糖体（较轻的结构）的存在。左下：核糖体的三维重建的表面表示，从B中所示的图像确定为18.9。改编自Lu等人。 J. struct。 Biol .. 参考： Barnard D和T Wagenknecht。 2005。用于冷冻电子显微镜中的毫秒时间分辨率的气动微喷枪。显微镜和微分析11：290-291。 Berriman J和N Unwin。 1994。通过冷冻微观镜结合喷雾液滴的快速混合对瞬态结构的分析。超镜检查56：241-252。 Subramaniam，S.，M。Gerstein，D。Oesterhelt和R. Henderson。 1993。对细菌紫红素光循环的结构变化的电子衍射分析。 Embo J. 12：1-8。 Tittor，J.，S。Paula，S。Subramaniam，J。Heberle，R。Henderson和D. Oesterhelt。 2002。在没有实质构象变化的情况下，细菌淡氨宁的质子易位。 J. Mol。生物。 319：555-565。 Unwin，N.1995。以开放状态成像的乙酰胆碱受体通道。自然373：37-43。 Walker，M.，X.-Z.Zhang，W.Jiang，J.Trinick和H.D. White。 1999年。通过停止流量荧光和毫秒时间分辨率的肌球蛋白亚纹理1与肌动蛋白结合的瞬态障碍的观察：肌肉中Crossbridge Power中风的开始具有可变的几何形状。 Proc。纳特。学院。科学。美国96：465-470。 White，H.D.，K。Thirumurugan，M.L。沃克和J. Trinick。 2003年。使用步进电动机和电喷雾的时间分辨电子冷冻微镜的第二代设备。 J. struct。生物。 144：246-252。 White，H.D.，M.L。沃克和J. Trinick。 1998年。用于毫秒时间分辨率电子显微镜检查的计算机控制的喷涂机器。 J. struct。生物。 121：306-313。 在上一个报告期间，介绍了以下摘要： + lu，Z.，McMahon，J.，Mohamed，H.，Barnard，D.，Shaikh，T.R。，Wagenknecht，T。和Lu，T.M。 2008。用于时间分辨的冷冻电子显微镜的微流体混合系统。显微镜和微分析14：1598-1599。 + Barnard，D.，Lu，Z.，Shaikh，T.R.，Mohamed，H.，Buttle，K.和Wagenknecht，T.2008。1576-POS开发反应混合器/微型神经纤维剂，用于时间分辨的大分子系统的冷冻电子显微镜。生物。 J. 94：1576。