我们感谢帮助提供植物行表示共振传感器与视频准备和Bhavna乔德赫瑞:菲利普Denninger。这项工作是由国家科学基金会（MCB 1021677），能源部（DE-FG02-04ER15542）WBF的，国家卫生研究院，霍华德·休斯医学研究所SRQGG赠款支持由欧洲分子生物学组织长期支持长期研究金。 MM的是由亚历山大·冯·洪堡基金会的支持。

注意:在无菌条件下执行的所有步骤的准备步骤。 1。塑料锥种子萌发的制备<ol><li>填写增长1％琼脂培养基厚度为5毫米，10厘米的培养皿。我们使用修改霍格兰德中等4半强度，但应选择培养基组成，以满足个人的实验要求。 </li><li>虽然仍然是液体介质，使用多通道移液器，以填补从培养皿中等5μL10μL枪头。 </li><li>存储在枪头中填充的提示，直到介质是固体，然后切成4毫米长的塑料锥和直立成一个Petri菜含有固体培养基中进行。 </li></ol> 2。种子萌发和幼苗生长的影响<ol><li>在5分钟的5％次氯酸钠表面消毒种子，用无菌水冲洗三次，然后将其放在一个单一的种子中充满锥顶第</li><li>密封微孔胶带（3M）和存储的菜，在4°C至同步萌发。 </li><li>三天后，转让板块增长内阁开始发芽。我们的生长条件是23°在16H高light/8h暗周期℃（光照强度:100μE米-2 S -1）。 </li><li> 5至7天发芽后，幼苗应该转移到RootChip准备。在这个时候，根尖应该是附近的塑料锥体底孔。在解剖显微镜下检查健康幼苗，根长，如果适用的话，表达了荧光标记。 </li><li>标记转移到芯片上的个别幼苗。选择十个左右的情况下一个苗在传输过程中被损坏。 </li></ol> 3。幼苗转移到RootChip <ol><li>消毒长期实验RootChip，包纸巾，在玻璃培养皿的地方，反应釜等设备。 </li><li>一旦冷却RootChip，包括液体培养基中。完全沉浸在RootChip应，但液面应不超过3毫米以上RootChip表面。 </li><li>与20μL移液器，拉根入口和室内插座通过媒介来填充介质的观察室。 </li><li>选择在步骤2.5到RootChip入口的插头塑料筒。锥体应紧贴在进气口。由于RootChip装上一层薄薄的光学玻璃，不要施加太大的压力芯片。 </li><li>在液体培养基中过夜孵育RootChip。为了防止浮动，在芯片上放置两个玻璃片。添加一个磁搅拌棒和关闭的菜。 </li><li>大会转移到磁力搅拌器轻轻搅动介质。 </li><li>该RootChip的入口在30°角相交通道设备正常FACIlitate根系生长，进入通道（ 图1A）。为了进一步支持增长所需的方向，稍微倾斜大会，网点芯片对面一侧玻片放置在培养皿上。 </li><li>为了保持光/暗周期，照亮苗用环形灯（光强度:100μE米-2 S -1），连接到一个计时器。 </li></ol> 4。连接的RootChip向承运人<ol><li>翌日，填补液体培养基中（ 图1B）一个密封pressurizable的小瓶。 </li><li>反转芯片载体，将其放置在一个稳定的表面。从液体介质RootChip删除和插入它的PDMS侧底部的芯片载体光圈。东方芯片，使含有控制层入口的一侧正面临载体侧壁的压力线在管接头一侧。 </li><li>干上的玻璃盖芯片的底部，用纸巾轻轻印迹。承运人的RootChip固定用胶带和权利的整体组装。 </li><li>管接头是由灵活的塑料微孔管（TYGON 0.20"内径x 0.060"外径）的切成5厘米长的块，并把它们连接到不锈钢微孔管（新英格兰小管，0.025"外径×0.013"编号×0.75"水长）。填写水使用注射器和芯片上相应的控制层入口插头每个油管接头油管接头。稍后将填补控制层通道和用于发射微机械阀门的压力。 </li><li>堵塞线到媒体/溶液小瓶（S）的两端。施加压力的溶液与空气的注射器小瓶。溶液小瓶内增加空气压力，将迫使该行的液体。 </li></ol> 5。安装在显微镜的RootChip <ol><li>将载体上的microscOPE阶段。为了减少装配了由于在房间的振动实验的过程中转移的可能性，承运人应符合完全进入的阶段插入槽口。 </li><li>由气压控制芯片的芯片阀门，并通过流动的介质。两行与监管机构的分支的主要压力线 - 一个用来控制通过介质流动的渠道，与其他连接到驱动控制层的推阀的电磁空气阀。从计算机通过USB阀门控制器（拉斐尔·戈麦斯舍贝里，劳伦斯伯克利国家实验室开发的），电磁阀的操作。之前关闭两个压力调节器连接芯片。 </li><li>承运人水库添加几毫升的水，保持湿度在大会高。这一步应重复了较长的实验过程中保持干燥的植物。保持较低的音量，尽量减少ţ他量的液体，可以洒在显微镜上。长期的实验，从芯片插座流出可以通过连接芯片网点，微孔管（见第4.4步）水库引导承运人的水库。另外，流出的芯片表面上的积累，可以收集吸取。 </li><li>准备从透明的塑料板保护（C线）的平方表。透明塑料双面胶带固定载体，以保持高湿度大会。 </li><li>定位在芯片的环形灯，并保持在光/暗周期。环形灯应关闭任何实验，使用荧光标记物，直接光照会干扰图像采集开始前。 </li></ol> 6。使用LabVIEW接口操作RootChip LabVIEW软件平台的RootChip控制器接口下载我们网站<a href="http://dpb.carnegiescience.edu/technology/rootchip" target="_blank">http://dpb.carnegiescience.edu/technology/rootchip</a> 。 <ol><li>芯片上的阀门关闭，施加压力，控制层，在这种情况下，通过开放的电磁空气阀。控制器的接口，可以通过点击下面的按钮阀门数量阀门驱动。明亮的绿色表示应用程序的压力，一个芯片上的截止阀（ 图2B）。激活开放前压力调节器的所有三个控制器接口解决方案进水阀。注:控制器接口设有一个反馈回路，从而使系统的状态监测。控制器界面中点击"回读"按钮，此功能可能会被激活。 </li><li>打开控制层的压力调节器和最初设置为15磅，然后打开流层中的稳压器，并初步设定为5磅。依赖丁所需的流量，压力可能会有所调整。 </li><li>打开进水阀的选择生长介质冲洗介质商会。 </li><li>检查在显微镜下的流动路径。通常情况下，空气中被困在流通渠道，必须拆除。此外，控制层的渠道仍然含有空气，必须逼出来的水从管接头（死胡同填充）所取代。这两项任务是实现由每八腔冲洗几次（5磅），直到所有的空气从被迫到的PDMS通道（"脱气"）。注:编程控制器接口，可自动化实验。这些例程也可用于到德加芯片。 </li></ol> 7。代表结果的RootChip的主要目的是结合的高集成度的成像平台和灌注系统在一个单一的设备。为了证明操纵我们的根微刷新暗食用色素（1:4在水耕介质稀释）商会和测量流体在商会的交流。在推荐的5磅的压力，我们测得的约1.5μL/分钟（ 图3）计算出的流量在10秒内充分交换。 我们还观察到幼苗根系生长，在这个生长在黑暗中，并与10 mM葡萄糖作为外部能量源（ 图4）提供的情况。根据介质的光组成的，如生长条件，植物可以观察在RootChip长达三天。 该RootChip已经被用来监测细胞内的葡萄糖和半乳糖水平表达基因编码的纳米传感器的根源，根据福斯特共振能量转移（FRET）5-7。在芯片的根部灌注与葡萄糖或半乳糖溶液的方形脉冲（ <strong图5）。细胞内糖的水平进行监测和显示在这里表示，作为承兑人荧光黄水晶的强度比捐助ECFP强度。中的比例上升，表明糖分积累。 <img src="/files/ftp_upload/4290/4290fig1.jpg" alt="图1" /> 图1。 RootChip原则 。 <ol class="ualpha"><li>该RootChip设有八个根系的生长和成像观察室。先发芽的种子，塑料锥 - 从塑料枪头制作 - 这是与固体介质填充。根尖生长中期和到达室连续流动的液体培养基中保持在室内恒定的条件。 （红色）的细观阀门控制流量。该芯片安装在光学玻璃盖。 图未按比例。 （改编格罗斯曼等人，2011年植物细胞的权限。） </li><li> SCH电磁辐射与膜片（红色）pressurizable溶液小瓶。 </li></ol><img src="/files/ftp_upload/4290/4290fig2.jpg" alt="图2" /> 图2。连接安装RootChip。 <ol class="ualpha"><li>安装在倒置显微镜完全的连接RootChip和载体的顶视图。 </li><li>计划说明系统的阀门和控制器接口。设置引导流体流动的一个单腔阀的例子所示。虽然阀门4至8单阀，阀门0至3组行为的行为。有了这个系统，通过激活一个阀门的组合可以解决个别室。 </li></ol><img src="/files/ftp_upload/4290/4290fig3.jpg" alt="图3" /> 图3。在观察CH交换的解决方案琥珀。交换使用染料溶液中观察室的流体可视化。图像是一个明亮的领域和假彩色染料的信号强度的叠加。 <img src="/files/ftp_upload/4290/4290fig4.jpg" alt="图4" /> 图4。葡萄糖/半乳糖在20H当然纳米传感器芯片上的根系生长。观察一个生长根表达了荧光共振。时间格式:HH:MM，比例尺:100微米。 <img src="/files/ftp_upload/4290/4290fig5.jpg" alt="图5" /> 图5。测量细胞内的血糖水平，使用共振的纳米传感器。 <ol class="ualpha"><li>量传感器显示为，茶晶内根尖强度（左）。细胞内的反应共振纳米传感器的葡萄糖或半乳糖溶液中的应用比例的图像显示。 （改编格罗斯曼等人的许可。，2011解放军NT细胞）比例尺:100微米。 </li><li>跟踪共振葡萄糖三个重复的方形脉冲（绿色）和半乳糖（红色）的响应比的变化。 </li></ol>

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没有利益冲突的声明。

的RootChip比传统的增长方式的主要优点是显微镜微创准备，可逆性和反复改变根环境的能力，和发展主管和生理健康组织的几天内连续观测的能力。此前，幼苗生长垂直上胶凝媒体和立即转移到灌注系统实验前，只允许测量单根时间8。微流控工具，已被用于拟南芥，但一体化水平低9或无灌注控制10。 RootChip结合的高集成度，能够自动通过精确的流量指导实验。这个平台上，所有11个微流体装置的特点，另一个优点是，只有极少量的液体需要提供必要的螺母根rients，甚至跨越数天的实验。 RootChip是目前设计为一个单一用途的设备，但因为芯片的生产成本低，消耗试剂少量使得芯片仍然很合算。 有一个必须采取的几个关键步骤，以保证幼苗的健康: 在塑料锥体的体积只有3-4微升，这将开始干燥时，暴露在空气中。因此它是至关重要的锥体转移到芯片快速和湿度保持高直到根部已经达到了观察室，这将提供足够的水。应迅速执行，并没有中断，以防止幼苗干燥步骤4.2至4.5。 3.5 - 3.8的步骤描述了芯片的液体介质，在此期间，根增长到观察室孵化。这一步可以跳过安装到C芯片arrier立即开始生长介质常数灌注。但是，我们建议在生长培养基浸泡过夜，因为它具有一些优点:1），它创建了一个潮湿的环境，使幼苗不太可能成为干，因为他们成长到观察室; 2）芯片被浸泡在液体中，所以脱气（步骤6.4）会更快。 重要的是要使用低溶质浓度媒体。较为集中的解决方案可能会沉淀，堵塞通道，特别是如果该芯片使用数天。 一旦设备连接到空气压线，流动的介质，通过改变阀门液压控制。为了保证适当的微机械阀门关闭，重要的是选择一个控制压力是比流压力高三倍左右。流压力应不超过15磅，根入口的流体将被推迟。较高的压力，马y也引起脱层的芯片，使芯片无法使用。 是硅橡胶是多孔性和疏水性的RootChip限制。虽然水溶液几乎是惰性材料，它可吸收有机物12。这可能会干扰有机化合物可能泄漏甚至当这种化合物的供应已经停止在进口材料的解决方案的快速交换。由于孔隙率，使用有机溶剂可能引起肿胀的PDMS 12。 我们将继续，优化的RootChip和作物根系，延长它的效用，例如。我们相信，通过改善获得治疗和观察根，像的RootChip微工具将打开根研究的新维度。

<table border="1"><tbody><tr><td> 项目 </td><td> 源 </td><td> 信息 </td></tr><tr><td>芯片载体，软件和其他信息。 </td><td>卡内基学会 - 存保</td><td> CAD和CNC文件为载体的制作，控制器软件和进一步的信息可以从网站上下载<a href="http://dpb.carnegiescience.edu/technology/rootchip" target="_blank">http://dpb.carnegiescience.edu/technology/rootchip</a>载体，也可以从这个网站订购。 </td></tr><tr><td> RootChip </td><td>斯坦福大学的铸造</td><td>光罩设计和制造协议应要求提供。现成使用RootChips，可从网上订购<a href="http://www.stanford.edu/group/foundry/" target="_blank">http://www.stanford.edu/group/foundry/</a> </td></tr><tr><td>芯片控制器</td><td> 家庭内置</td><td>最初是由拉斐尔·戈麦斯舍贝里，劳伦斯伯克利国家实验室的自动化阀门控制器系统。一个详细的说明如何建立你自己的驱动阀门控制器，可以发现在<a href="https://sites.google.com/a/lbl.gov/microfluidics-lab/valve-controllers" target="_blank">https://sites.google.com/a/lbl.gov/microfluidics-lab/valve-controllers</a> </td></tr></tbody></table>

time-lapse fluorescence imaging of arabidopsis root growth with rapid manipulation of the root environment using the rootchip

作为植物的物理锚根职能和机关负责，如氮，磷，硫和微量元素，植物从土壤中获得水分和矿质营养的吸收。如果我们要发展可持续的方法来生产作物产量高，我们需要更好地了解如何根的发展，需要的营养物质很宽的频谱，共生和病原微生物的相互作用。为了实现这些目标，我们需要能够探索随着时间的推移时间从几分钟到几天不等的根在微观细节。 我们开发的RootChip，聚二甲基硅氧烷（PDMS）中-基于微流体装置，它允许我们成长，从拟南芥的形象根，同时避免任何生理上的压力，在制备过程中，根1（ 图1）成像。该器件包含一个岔渠道结构，引导流体流动的细观阀门从解决方案入口每8个观察室2。这灌注系统允许根微环境进行控制和修改的精度和速度。室的体积大约是400 NL，因此要求只有极少量的测试解决方案。 在这里，我们提供了一个使用实时分辨率成像为基础的方法RootChip根生物学研究的详细协议。根可超过数天使用时间的推移显微镜分析。根源可以灌注营养液抑制剂，可以并行分析多达八个幼苗。该系统具有潜在的应用范围很广，包括化学品，荧光为基础的基因表达分析，生物传感器分析存在或不存在的情况下在根系生长的分析，如共振的纳米传感器3。

Watch this Scientific Journal Video about Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip at JoVE.com

Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip

本文提供中的RootChip的拟南芥幼苗栽培的协议，一个微成像平台，结合微观根监测和基于FRET的细胞代谢水平的测量与自动化控制生长条件。

时间推移荧光成像拟南芥根系生长的根环境的快速操作使用RootChip

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, ...

time-lapse-fluorescence-imaging-arabidopsis-root-growth-with-rapid

Research

JoVE Journal

Bioengineering

19.6K 次观看.  Carnegie Institution for Science. 本文提供中的RootChip的拟南芥幼苗栽培的协议，一个微成像平台，结合微观根监测和基于FRET的细胞代谢水平的测量与自动化控制生长条件。

视频: Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip

Demonstrating the Uses of the Novel Gravitational Force Spectrometer to Stretch and Measure Fibrous Proteins

The study of macromolecular structure has become critical to the elucidation of molecular mechanisms and function. There are several limited, but important bioinstruments capable of testing the force dependence of structural features in proteins. Scale has been a limiting parameter on how accurately researchers can peer into the nanomechanical world of molecules, such as nucleic acids, enzymes, and motor proteins that perform life-sustaining work. Atomic force microscopy (AFM) is well tuned to determine native structures of fibrous proteins with a distance resolution on par with electron microscopy. However, in AFM force studies, the forces are typically much higher than a single molecule might experience 1, 2. Optical traps (OT) are very good at determining the relative distance between the trapped beads and they can impart very small forces 3. However, they do not yield accurate absolute lengths of the molecules under study. Molecular simulations provide supportive information to such experiments, but are limited in the ability to handle the same large molecular sizes, long time frames, and convince some researchers in the absence of other supporting evidence2, 4.
The gravitational force spectrometer (GFS) fills a critical niche in the arsenal of an investigator by providing a unique combination of abilities. This instrument is capable of generating forces typically with 98% or better accuracy from the femtonewton range to the nanonewton range. The distance measurements currently are capable of resolving the absolute molecular length down to five nanometers, and relative bead pair separation distances with a precision similar to an optical trap. Also, the GFS can determine stretching or uncoiling where the force is near equilibrium, or provide a graded force to juxtapose against any measured structural changes. It is even possible to determine how many amino acid residues are involved in uncoiling events under physiological force loads 2. Unlike in other methods where there is extensive force calibration that must precede any assay, the GFS requires no such force calibration 5. By complementing the strengths of other methods, the GFS will bridge gaps in understanding the nanomechanics of vital proteins and other macromolecules.

This is a step-by step guide showing the purpose, operation, and representative results from the novel gravitational force spectrometer.

展示新型引力光谱仪的使用拉伸和测量纤维蛋白

The study of macromolecular structure has become critical to the elucidation of molecular mechanisms and function. There are several limited, but ...

科研

生物工程

Axon Stretch Growth: The Mechanotransduction of Neuronal Growth

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata are separated from their axon terminals due to skeletal growth of the enlarging organism (Weiss 1941; Gray, Hukkanen et al. 1992). This mechanotransduction induces a secondary mode of neuronal growth capable of accommodating continual elongation of the axon (Bray 1984; Heidemann and Buxbaum 1994; Heidemann, Lamoureux et al. 1995; Pfister, Iwata et al. 2004).
Axon Stretch Growth (ASG) is conceivably a central factor in the maturation of short embryonic processes into the long nerves and white matter tracts characteristic of the adult nervous system. To study ASG in vitro, we engineered bioreactors to apply tension to the short axonal processes of neuronal cultures (Loverde, Ozoka et al. 2011). Here, we detail the methods we use to prepare bioreactors and conduct ASG. First, within each stretching lane of the bioreactor, neurons are plated upon a micro-manipulated towing substrate. Next, neurons regenerate their axonal processes, via growth cone extension, onto a stationary substrate. Finally, stretch growth is performed by towing the plated cell bodies away from the axon terminals adhered to the stationary substrate; recapitulating skeletal growth after growth cone extension.
Previous work has shown that ASG of embryonic rat dorsal root ganglia neurons are capable of unprecedented growth rates up to 10mm/day, reaching lengths of up to 10cm; while concurrently resulting in increased axonal diameters (Smith, Wolf et al. 2001; Pfister, Iwata et al. 2004; Pfister, Bonislawski et al. 2006; Pfister, Iwata et al. 2006; Smith 2009). This is in dramatic contrast to regenerative growth cone extension (in absence of mechanical stimuli) where growth rates average 1mm/day with successful regeneration limited to lengths of less than 3cm (Fu and Gordon 1997; Pfister, Gordon et al. 2011). Accordingly, further study of ASG may help to reveal dysregulated growth mechanisms that limit regeneration in the absence of mechanical stimuli.

A unique tissue engineering method was developed to elongate numerous nerve fibers in culture by recapitulating axon stretch growth; a form of nervous system growth whereby nerves elongate in conjunction with growth of the enlarging body.

轴突伸展生长:神经生长Mechanotransduction

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata ...

Gold Nanostar Synthesis with a Silver Seed Mediated Growth Method

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest in using nano-colloids for photo-thermal ablation, drug delivery and many other biomedical applications 6. Gold is particularly used because of its low toxicity 7-9. A property of metal nano-colloids is that they can have a strong surface plasmon resonance 10. The peak of the surface plasmon resonance mode depends on the structure and composition of the metal nano-colloids. Since the surface plasmon resonance mode is stimulated with light there is a need to have the peak absorbance in the near infrared where biological tissue transmissivity is maximal 11, 12.
We present a method to synthesize star shaped colloidal gold, also known as star shaped nanoparticles 13-15 or nanostars 16. This method is based on a solution containing silver seeds that are used as the nucleating agent for anisotropic growth of gold colloids 17-22. Scanning electron microscopy (SEM) analysis of the resulting gold colloid showed that 70 % of the nanostructures were nanostars. The other 30 % of the particles were amorphous clusters of decahedra and rhomboids. The absorbance peak of the nanostars was detected to be in the near infrared (840 nm). Thus, our method produces gold nanostars suitable for biomedical applications, particularly for photo-thermal ablation.

We synthesized star shaped gold nanostars using a silver seed mediated growth method. The diameter of the nanostars ranges from 200 to 300 nm and the number of tips vary from 7 to 10. The nanoparticles have a broad surface plasmon resonance mode centered in the near infrared.

黄金Nanostar合成银种子介导的生长方法

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

创建的胶粘剂和可溶性梯度荧光显微镜成像细胞迁移

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Sample Drift Correction Following 4D Confocal Time-lapse Imaging

The generation of four-dimensional (4D) confocal datasets; consisting of 3D image sequences over time; provides an excellent methodology to capture cellular behaviors involved in developmental processes.&#160; The ability to track and follow cell movements is limited by sample movements that occur due to drift of the sample or, in some cases, growth during image acquisition. Tracking cells in datasets affected by drift and/or growth will incorporate these movements into any analysis of cell position. This may result in the apparent movement of static structures within the sample. Therefore prior to cell tracking, any sample drift should be corrected. Using the open source Fiji distribution 1 &#160;of ImageJ 2,3 and the incorporated LOCI tools 4, we developed the Correct 3D drift plug-in to remove erroneous sample movement in confocal datasets. This protocol effectively compensates for sample translation or alterations in focal position by utilizing phase correlation to register each time-point of a four-dimensional confocal datasets while maintaining the ability to visualize and measure cell movements over extended time-lapse experiments.

Time-lapse microscopy allows the visualization of developmental processes. Growth or drift of samples during image acquisition reduces the ability to accurately follow and measure cell movements during development. We describe the use of open source image processing software to correct for three dimensional sample drift over time.

样品漂移校正继4D共焦时间推移成像

The generation of four-dimensional (4D) confocal datasets; consisting of 3D image sequences over time; provides an excellent methodology to capture ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

3d轨道跟踪的改进双光子显微镜之应用:细胞内的囊泡的跟踪

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

Root hairs increase root surface area for better water uptake and nutrient absorption by the plant. Because they are small in size and often obscured by their natural environment, root hair morphology and function are difficult to study and often excluded from plant research. In recent years, microfluidic platforms have offered a way to visualize root systems at high resolution without disturbing the roots during transfer to an imaging system. The microfluidic platform presented here builds on previous plant-on-a-chip research by incorporating a two-layer device to confine the Arabidopsis thaliana main root to the same optical plane as the root hairs. This design enables the quantification of root hairs on a cellular and organelle level and also prevents z-axis drifting during the addition of experimental treatments. We describe how to store the devices in a contained and hydrated environment, without the need for fluidic pumps, while maintaining a gnotobiotic environment for the seedling. After the optical imaging experiment, the device may be disassembled and used as a substrate for atomic force or scanning electron microscopy while keeping fine root structures intact.

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

This article demonstrates how to culture Arabidopsis thaliana seedlings in a two-layer microfluidic platform that confines the main root and root hairs to a single optical plane. This platform can be used for real-time optical imaging of fine root morphology as well as for high-resolution imaging by other means.

影像学在两层微流控平台拟南芥幼苗的根头发形态

Root hairs increase root surface area for better water uptake and nutrient absorption by the plant. Because they are small in size and often obscured by ...

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to conventional epi-fluorescence microscopy, various imaging techniques have been developed to achieve specific experimental goals. Some of the widely used techniques include single-molecule fluorescence resonance energy transfer (smFRET), which can report conformational changes and molecular interactions with angstrom resolution, and single-molecule detection-based super-resolution (SR) imaging, which can enhance the spatial resolution approximately ten&#160;to twentyfold compared to diffraction-limited microscopy. Here we present a customer-designed integrated system, which merges multiple imaging methods in one microscope, including conventional epi-fluorescent imaging, single-molecule detection-based SR imaging, and multi-color single-molecule detection, including smFRET imaging. Different imaging methods can be achieved easily and reproducibly by switching optical elements. This set-up is easy to adopt by any research laboratory in biological sciences with a need for routine and diverse imaging experiments at a reduced cost and space relative to building separate microscopes for individual purposes.

Here we present a practical guide of building an integrated microscopy system, which merges conventional epi-fluorescent imaging, single-molecule detection-based super-resolution imaging, and multi-color single-molecule detection, including single-molecule fluorescence resonance energy transfer imaging, into one set-up in a cost-efficient way.

使用一个荧光显微镜进行多种成像模式

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to ...

Biomimetic Replication of Root Surface Microstructure using Alteration of Soft Lithography

Biomimetics is the use of chemistry and material sciences to mimic biological systems, specifically biological structures, to better humankind. Recently, biomimetic surfaces mimicking the microstructure of leaf surface, were used to study the effects of leaf microstructure on leaf-environment interactions. However, no such tool exists for roots. We developed a tool allowing the synthetic mimicry of the root surface microstructure into an artificial surface. We relied on the soft lithography method, known for leaf surface microstructure replication, using a two-step process. The first step is the more challenging one as it involves the biological tissue. Here, we used a different polymer and curing strategy, relying on the strong, rigid, polyurethane, cured by UV for the root molding. This allowed us to achieve a reliable negative image of the root surface microstructure including the delicate, challenging features such as root hairs. We then used this negative image as a template to achieve the root surface microstructure replication using both the well-established polydimethyl siloxane (PDMS) as well as a cellulose derivative, ethyl cellulose, which represents a closer mimic of the root and which can also be degraded by cellulase enzymes secreted by microorganisms. This newly formed platform can be used to study the microstructural effects of the surface in root-microorganism interactions in a similar manner to what has previously been shown in leaves. Additionally, the system enables us to track the microorganism&#8217;s locations, relative to surface features, and in the future its activity, in the form of cellulase secretion.

Biomimetics has been previously used as a tool to study leaf-microorganism interactions. However, no such tool exists for roots. Here, we develop a protocol to form synthetic surfaces mimicking root surface microstructure for the study of root-environment interactions.

利用软光刻改变根面微结构的仿生复制

Biomimetics is the use of chemistry and material sciences to mimic biological systems, specifically biological structures, to better humankind. Recently, ...

Blood Flow Imaging with Ultrafast Doppler

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler modes, it has several limits. Firstly, a finely tuned signal filtering operation is needed to distinguish blood flows from surrounding moving tissues. Secondly, the operator must choose between localizing the blood flows or quantifying them. In the last two decades, ultrasound imaging has undergone a paradigm shift with the emergence of ultrafast ultrasound using unfocused waves. In addition to a hundredfold increase in framerate (up to 10000 Hz), this new technique also breaks the conventional quantification/localization trade-off, offering a complete blood flow mapping of the field of view and a simultaneous access to fine velocities measurements at the single-pixel level (down to 50 &#181;m). This data continuity in both spatial and temporal dimensions strongly improves the tissue/blood filtering process, which results in an increase sensitivity to small blood flow velocities (down to 1 mm/s). In this method paper, we aim to introduce the concept of ultrafast Doppler as well as its main parameters. Firstly, we summarize the physical principles of unfocused wave imaging. Then, we present the Doppler signal processing main steps. Particularly, we explain the practical implementation of the critical tissue/blood flow separation algorithms and on the extraction of velocities from these filtered data. This theoretical description is supplemented by in vitro experiences. A tissue phantom embedding a canal with flowing blood-mimicking fluid is imaged with a research programmable ultrasound system. A blood flow image is obtained and the flow characteristics are displayed for several pixels in the canal. Finally, a review of in vivo applications is proposed, showing examples in several organs such as carotids, kidney, thyroid, brain and heart.

This protocol shows how to apply ultrafast ultrasound Doppler imaging to quantify blood flows. After a 1 s long acquisition, the experimenter has access to a movie of the full field of view with axial velocity values for each pixel every &#8776;0.3 ms (depending on the ultrasound time of flight).

血流成像与超快多普勒

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler ...