我们感谢Tzlil Rozenblat，亚历山德拉·贝洛和卡尔·Spuhler的技术援助。这项工作是由瓦瑟学院本科生科研暑期学院（URSI），露西·梅纳德·鲑鱼研究基金和美国航空航天局奖＃NX09AU90A，国家科学基金会（National Science Foundation）卓越研究中心科学与技术奖（NSF-CREST）＃0630388和美国国家科学基金会奖＃1058385。

1。C.线虫的准备工作视频分析<ol><li>将10-20妊娠的成年线虫新鲜的NGM琼脂填充的Petri板使用薄，扁平铂金线挑。充满的NGM琼脂的培养皿中包含一个小的圆形光斑，E.大肠杆菌 （增长有限应变，OP50，给出了最好的结果）的线虫吃。允许的线虫产卵3-5小时，然后取出大人，从而建立了一个小的，同步发展的文化50-150线虫。允许线虫增长到成年早期的Petri板在20℃下孵育4-5天。这些培养方法是基于既定的程序（Stiernagle 2006）5 </li><li>在当天的视频分析，年轻的成年线虫1毫升去离子水，蒸馏水冲洗板，收集50-150蠕虫，从同步的文化。类似M9或PBS缓冲溶液也可以被使用。使用的离心旋转的w奥姆斯管的底部，或允许他们重力沉降约15分钟。从管中移除大部分的水，并替换它与1毫升去离子水，蒸馏水，为了洗掉从线虫任何粘附的细菌。 </li><li>将在水或缓冲液的石英比色皿，用微量的线虫。重要的是要与一个小数目，甚至更少的在同一时间或约5-10线虫数量，以便在一个时间的一个线虫是可能要由激光照射。如果在上的培养板的线虫的数目太大，稀释在水或缓冲液中的蠕虫，直到达到所希望的密度。另外，也可以挑选个别线虫一滴上新鲜的琼脂平板上的水或缓冲液洗并转移到比色皿的个别蠕虫。在我们的研究中，我们使用1毫米，2毫米和5毫米比色皿尺寸检查的物理限制的游泳行为的影响。 </li></ol> 2。光学视频Analysi设置小号<ol><li>使用543 nm（绿色）氦氖（氦氖）激光，两个前表面铝镜，一个小杯保持器，一投影屏幕上，和一个高速的摄像头（卡西欧高速卡西欧），能够执行安装，如在图1中示出〜240 fps的拍摄。两个反光镜被用于引导的激光束，通过比色皿中。可以使用不同尺寸的比色皿，根据性质的研究。作为一个例子，我们使用的比色皿的5毫米厚的游泳频率显示的空间约束的影响。的激光束必须满足要求的过采样， 即 ，生物体应阻碍的激光束的不超过三分之一。对于C。线虫 ，激光束应该是大约2毫米的直径，当它与线虫交互。的激光束应不大于5毫米的直径，因为衍射图案将难以定位。在屏幕上的衍射机体的距离要大得多机体本身。 </li><li>将石蜡膜的比色皿的顶部上，并倒转混合线虫到水柱。放置，使比色皿中​​的比色皿支架的蠕虫是最初的比色皿的顶部附近。使用反射镜时，引导的激光束，通过该中心的比色皿中。由于线虫的密度比水的，它们会慢慢下降的比色皿的底部，而在相同的时间内的水柱游泳。 </li><li>在投影屏幕上，彩色的点，对应于所发送的激光束黑，以减少散射作为发送波束满足投影屏幕上（ 图2）。或者把在投影屏幕上的开口，以允许的发射光束通过屏幕。消除或减少的发射光束的散射，将保持饱和由于发送波束的相机的CCD阵列。 </li><li>要捕获的衍射图案的大小的度量，画一条线约5厘米，在投影屏幕上不干扰的衍射光图像的情况下所发送的激光束光斑的旁边。 </li><li>开始用相机记录房间的灯是5厘米的线段是在同一个镜头的衍射图像。打开房间的灯关闭。记录衍射图像在屏幕上的摄像头，如蠕虫通过激光束。 </li></ol> 3。视频数据准备<ol><li>在电脑上安装视频分析程序（记录仪专业版： <a href="http://www.vernier.com/products/software/lp/" target="_blank">http://www.vernier.com/products/software/lp/</a> ）。将视频导入到视频分析程序</li><li>设置原点，以配合所发射的激光光斑。设置使用在5厘米线的规模，在投影屏幕上。 </li><li>追踪每个使用的视频分析软件的线虫所形成的衍射图像（ 图3）的角位移</li><li>要找到安古拉ŕ位移的反正切（Y / X），复制和粘贴数据到电子表格（Excel）和180°或π的所有负面的角度来产生连续的曲线（Figure. 4）。 </li></ol> 4。即时观察游泳运动频率的实时数据采集<ol><li>使用相同的设置为视频分析，放置一个光电二极管（DET10A来自Thorlabs公司）与偏离中心的小区域（〜1毫米2）中的衍射图案，在投影屏幕的右前方。 </li><li>数字示波器（由笔克科技的PicoScope）连接到电脑通过USB端口连接的光电二极管。注意在计算机屏幕上的颠簸模式。 </li><li>在ASCII文本格式保存采集到的数据集。 </li></ol> 5。数据分析<ol><li>导入采集的数据使用视频或光电二极管的拟合波形数据分析程序，能够用卡方最小化。这里使用的程序是地（ <a href="http://www.originlab.com/" target="_blank">http://www.originlab.com/</a> ），。合身的正弦曲线，以确定抖动频率（ 图4和图5）。 </li><li>平均频率从各种样品的游泳池，并确定变异。在这项研究中，数据进行统计学分析使用单因素方差分析，接着用Bonferroni多重比较测试。 p值&lt;0.05为差异有统计学意义。 </li></ol> 6。使用数学模型衍射图样为例 注：可以使用许多不同的计算工具来模拟衍射图案。此过程会有所不同不同的计算工具，如MATLAB，Excel中，原产地等。 <ol><li> 创建映像：显微镜载片上，使用传统的显微镜拍摄的线虫。 </li><li> 二值化图像导入蠕虫图像的蠕虫“我置身于数学的图像拖动到Mathematica。将图像转换成黑白图像（二值化）。这样就可以实现“二值化”命令，在Mathematica：BlackandWhiteImage二值化蠕虫。然后，该图像可以被视为一个黑色和白色图像。 另外，使用“EdgeDetect”命令来创建一个BlackandWhiteImage：EdgeDetect蠕虫，6.0,0.035]，其中两个尾随的数字，控制粗糙的边缘。 </li><li> 转换图像转换成0和1的矩阵：这样就可以实现在Mathematica“的ImageData”命令：矩阵的ImageData [BlackandWhiteImage]。 </li><li> 傅立叶变换矩阵：在Mathematica中使用&#39;傅立叶命令进行傅立叶变换的矩阵使用指定的FourierParameters：FTransform傅立叶矩阵，FourierParameters - &gt; {0，-2}]。 </li><li>广场的绝对模平方矩阵得到的的衍射矩阵和改善图像的对比度：值矩阵，并查看所得到的图像，这是对应于原始图像的衍射图案。此外，使用“日志”功能来缩放图像的对比度：ImageContrast图片[登录[1 +（ABS ​​[FTransform]）^ 2] / 0.5]。 </li><li> 调整图像大小，方便查看：作物中央衍射图案调整为所需的ImageResize ImageContrast [Y]，100，500]。 </li><li> 比较建模的衍射图案与衍射图案获得自由游动的蠕虫。 （图6） </li></ol>

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没有利益冲突的声明。

我们已经开发出一种新颖的方法来运动的实时测量和简单的运动器官的行为在微观有机体如线虫，不需要使用显微镜。8这种方法的方法也可以用于研究许多微生物，如原生生物。此方法只限于所使用的光的波长。的生物体不应该是小于光的波长。除了所需要的设备的成本节省和便携性，这种方法的一个主要优点是在实时和在三个维度的行为的能力来衡量，在显微镜下的图像平面没有狭窄的限制。它也可以使用这种技术，研究重力或许多其他条件的行为，可以不使用显微镜为基础的方法进行研究的影响。因此，我们可以更好地了解微生物的自然运动器官BEHAviors从显微镜载玻片上滴或专门的微流体室（Park 等 ，2008年）的限制中解脱出来。 的衍射图案中的相位信息的缺乏不允许衍射自远场衍射图案的傅立叶变换的绝对值的平方成正比的对象相对应的图像的直接检索。因此，我们现蠕虫图像计算衍射图案，使他们能够自由游泳线虫（ 图6）与衍射图案的匹配。 这种方法已经取得了阶段性成果，为真正的自由游泳C.线虫，并可以应用到任何微观物种回旋像水或许多不同的离子溶液中的光学透明的环境。只允许常规显微镜研究微米的顺序上与焦点深度11，这是由于有限的景深时的光线聚焦： <img alt="式（1）" src="/files/ftp_upload/4412/4412eq1.jpg" /> 这里的F-数N有互惠关系的混乱（三）与圆 ，短焦距与大C。12,13衍射法虽然这肯定不是一个替代传统的显微镜，它能够很快，所以甚至可以被操纵，物种实时低成本提供定量结果。的衍射图案，可以得到与任何激光指针。可以以较低的时间分辨率，使用普通的数码相机拍摄的衍射图案。虽然用户可能没有随时可用显微镜或光电二极管，在此实验中，如测量抖动的频率和评价衍射图案的关键部位，可以以非常低的成本完成。

<table border="1"><tbody><tr><td> 名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> 543 nm的氦氖激光</td><td> Melles Griot公司</td><td> LGX1 </td><td>在可见光范围内具有小于5 mW任何激光都可以使用。 </td></tr><tr><td> 2个前表面铝镜</td><td> Thorlabs公司</td><td> PF10-03-F01 </td><td></td></tr><tr><td>高速EXILIM相机</td><td>卡西欧</td><td></td><td></td></tr><tr><td>石英比色皿</td><td> Starna细胞</td><td> 21/G/5 </td><td></td></tr><tr><td> LoggerPro（软件） </td><td>游标</td><td></td><td> <a href="http://www.vernier.com/products/software/lp/" target="_blank">http://www.vernier.com/products/software/lp/</a> </td></tr><tr><td>数学8 </td><td>钨</td><td></td><tD&gt; <a href="http://www.wolfram.com/" target="_blank">http://www.wolfram.com/</a> </td></tr></tbody></table>

quantitative locomotion study of freely swimming micro-organisms using laser diffraction

土壤和水生微生物的生活和行为在一个复杂的三维环境。相反，大多数微小的有机体行为的研究，已用显微镜为基础的方法，极限的运动和行为，近一个狭窄的二维重点领域。我们提出了一种新的分析方法，提供实时分析自由游泳C.而不依赖于显微镜为基础的设备中的一个反应杯中线虫 。这种方法包括跟踪引导激光通过比色皿的光所产生的时间的周期性的X射线衍射图案。我们测量振荡频率为自由游动的线虫。 远场衍射图案的分析揭示了有关波形的线虫的线索。衍射是弯曲物体周围的光的过程。在这种情况下的光被衍射的生物体。光波的干涉，并可以形成广告也是衍射极限的格局。一种远场，或夫琅和费衍射图案形成，如果屏幕到对象的距离为远远大于衍射对象。在这种情况下，衍射图案可以计算（模拟）使用傅里叶变换。 是自由生活的线虫生活在土壤中的线虫，在三维空间导航。他们移动的正弦运动器官的模式称为爬行，在不同的模式称为游泳液体在固体基质，如土壤或琼脂。3扮演的角色的感官信息提供mechanosensory，化学感受，thermosensory的细胞，支配塑料运动器官的变化模式和开关模式才刚刚开始被阐明。我们描述一个光学测量线虫运动在三个方面，不需要显微镜，让我们开始探索线虫运动的复杂性，根据不同的合作方式条件。

<html>作为一个例子，我们研究了C.线虫在石英比色皿1厘米宽，5毫米厚，4厘米高的比色皿。采样一个单一的蠕虫使用视频分析，从视频分析获得的平均游泳频率在5毫米厚的比色皿是大约2.5赫兹的（ 图4）。同样，采样蠕虫病毒利用一个单一的实时数据采集方法，我们得到一个游泳池，频率约为2.7赫兹（ 图5），使用数字示波器（PicoScope软件），这个过程可以重复许多蠕虫。自由游泳的蠕虫的详细研究表明平均游泳频率2.37赫兹5毫米比色皿中。6正如预期的那样，在游泳池频率是高于爬行蠕虫（〜0.8赫兹）。3使用该衍射方法，平均游泳的频率是 C。 线虫 ，这仅限于显微镜载玻片上，已经发现，2 Hz的频率相匹配的原样的值。1,7 ve_content“&gt;以下程序3），然后6）允许建模游泳衍射图案，与用常规的显微镜获得的图像的蠕虫的帮助。建模的衍射图案是用来模拟的线虫游泳周期（实际可行的连续游泳游泳的频率相匹配的模式，由图6）。一个成功的典范。该蠕虫病毒应该是在游泳周期结束时相同的形状，因为它是在游泳周期的开始。 <img alt="图1" src="/files/ftp_upload/4412/4412fig1.jpg" /> 图1。一个绿色的氦氖激光被用来创建一个的动态衍射图案采用现场C.线虫 。衍射图谱在240 fps的拍摄。 <img alt="图2" src="/files/ftp_upload/4412/4412fig2.jpg" /> 图2绘制直线<html>翼一个黑点增加的发射光束的吸收。相机由于散射光的饱和性被降低，变得可见的衍射图案。 <img alt="图3" src="/files/ftp_upload/4412/4412fig3.jpg" /> 图3。屏幕截图的视频分析软件（记录仪专业版）的蠕虫被跟踪的衍射图案。 <a href="http://www.jove.com/files/ftp_upload/4412/4412fig3large.jpg" target="_blank">点击此处查看大图</a> 。 <img alt="图4" src="/files/ftp_upload/4412/4412fig4.jpg" /> 图4的视频数据对应于5毫米比色皿中的线虫游泳周期。曲线拟合揭示了游泳〜2.5 Hz的频率。 “/&gt; 图5。实时数据对应于5毫米比色皿中的线虫游泳周期。曲线拟合揭示了游泳〜2.7 Hz的频率。 <img alt="图6" src="/files/ftp_upload/4412/4412fig6.jpg" /> 图6的最上面一行表示实际的衍射图案和相匹配的建模的底部行中的衍射图案。使用显微镜载片上（中间行）的蠕虫生产建模的衍射图案。

Watch this Scientific Journal Video about Quantitative Locomotion Study of Freely Swimming Micro-organisms Using Laser Diffraction at JoVE.com

Quantitative Locomotion Study of Freely Swimming Micro-organisms Using Laser Diffraction

像自由游动的线虫的微生物<em&gt; C.线虫</em&gt;，生活和行为在一个复杂的三维环境。我们提供一个新的方法，分析报告<em&gt; C.线虫</em&gt;用衍射图案。这种方法包括跟踪的时间周期的衍射图案所产生的激光通过试管指导。

使用激光衍射自由游泳微生物的定量步态研究

Soil and aquatic microscopic organisms live and behave in a complex three-dimensional environment. Most studies of microscopic organism behavior, in ...

quantitative-locomotion-study-freely-swimming-micro-organisms-using

Research

JoVE Journal

Bioengineering

11.5K 次观看.  Vassar College. 像自由游动的线虫的微生物 C.线虫，生活和行为在一个复杂的三维环境。我们提供一个新的方法，分析报告 C.线虫用衍射图案。这种方法包括跟踪的时间周期的衍射图案所产生的激光通过试管指导。

视频: Quantitative Locomotion Study of Freely Swimming Micro-organisms Using Laser Diffraction

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck 1-2.
To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.

改进的可视化和定量分析，药物使用Micropatterned细胞的影响

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

科研

生物工程

Constructing a Low-budget Laser Axotomy System to Study Axon Regeneration in C. elegans

Laser axotomy followed by time-lapse microscopy is a sensitive assay for axon regeneration phenotypes in C. elegans1. The main difficulty of this assay is the perceived cost ($25-100K) and technical expertise required for implementing a laser ablation system2,3. However, solid-state pulse lasers of modest costs (&lt;$10K) can provide robust performance for laser ablation in transparent preparations where target axons are "close" to the tissue surface. Construction and alignment of a system can be accomplished in a day. The optical path provided by light from the focused condenser to the ablation laser provides a convenient alignment guide. An intermediate module with all optics removed can be dedicated to the ablation laser and assures that no optical elements need be moved during a laser ablation session. A dichroic in the intermediate module allows simultaneous imaging and laser ablation. Centering the laser beam to the outgoing beam from the focused microscope condenser lens guides the initial alignment of the system. A variety of lenses are used to condition and expand the laser beam to fill the back aperture of the chosen objective lens. Final alignment and testing is performed with a front surface mirrored glass slide target. Laser power is adjusted to give a minimum size ablation spot (&lt;1um). The ablation spot is centered with fine adjustments of the last kinematically mounted mirror to cross hairs fixed in the imaging window. Laser power for axotomy will be approximately 10X higher than needed for the minimum ablation spot on the target slide (this may vary with the target you use). Worms can be immobilized for laser axotomy and time-lapse imaging by mounting on agarose pads (or in microfluidic chambers4). Agarose pads are easily made with 10% agarose in balanced saline melted in a microwave. A drop of molten agarose is placed on a glass slide and flattened with another glass slide into a pad approximately 200 um thick (a single layer of time tape on adjacent slides is used as a spacer). A "Sharpie" cap is used to cut out a uniformed diameter circular pad of 13mm. Anesthetic (1ul Muscimol 20mM) and Microspheres (Chris Fang-Yen personal communication) (1ul 2.65% Polystyrene 0.1 um in water) are added to the center of the pad followed by 3-5 worms oriented so they are lying on their left sides. A glass coverslip is applied and then Vaseline is used to seal the coverslip and prevent evaporation of the sample.

Constructing a Low-budget Laser Axotomy System to Study Axon Regeneration in C. elegans

Laser axotomy followed by time-lapse imaging is a sensitive way to assay the effects of mutations in C. elegans on axon regeneration. A high quality, but inexpensive, laser ablation system can be easily added to most microscopes. Time lapse imaging over 15 hours requires careful immobilization of the worm.

构建一个低预算的激光干切断系统研究轴突再生 C。线虫</em

Laser axotomy followed by time-lapse microscopy is a sensitive assay for axon regeneration phenotypes in C. elegans1.  The main difficulty of this assay ...

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and pathways as well as its ease of cultivation. Several C. elegans disease models have been reported, including neurodegenerative disorders such as Parkinson's disease (PD), which involves the degeneration of dopaminergic (DA) neurons 1. Both transgenes and neurotoxic chemicals have been used to induce DA neurodegeneration and consequent movement defects in worms, allowing for investigations into the basis of neurodegeneration and screens for neuroprotective genes and compounds 2,3.
Screens in lower eukaryotes like C. elegans provide an efficient and economical means to identify compounds and genes affecting neuronal signaling. Conventional screens are typically performed manually and scored by visual inspection; consequently, they are time-consuming and prone to human errors. Additionally, most focus on cellular level analysis while ignoring locomotion, which is an especially important parameter for movement disorders.
We have developed a novel microfluidic screening system (Figure 1) that controls and quantifies C. elegans' locomotion using electric field stimuli inside microchannels. We have shown that a Direct Current (DC) field can robustly induce on-demand locomotion towards the cathode ("electrotaxis") 4. Reversing the field's polarity causes the worm to quickly reverse its direction as well. We have also shown that defects in dopaminergic and other sensory neurons alter the swimming response 5. Therefore, abnormalities in neuronal signaling can be determined using locomotion as a read-out. The movement response can be accurately quantified using a range of parameters such as swimming speed, body bending frequency and reversal time.
Our work has revealed that the electrotactic response varies with age. Specifically, young adults respond to a lower range of electric fields and move faster compared to larvae 4. These findings led us to design a new microfluidic device to passively sort worms by age and phenotype 6.
We have also tested the response of worms to pulsed DC and Alternating Current (AC) electric fields. Pulsed DC fields of various duty cycles effectively generated electrotaxis in both C. elegans and its cousin C. briggsae 7. In another experiment, symmetrical AC fields with frequencies ranging from 1 Hz to 3 KHz immobilized worms inside the channel 8.
Implementation of the electric field in a microfluidic environment enables rapid and automated execution of the electrotaxis assay. This approach promises to facilitate high-throughput genetic and chemical screens for factors affecting neuronal function and viability.

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans&#039; Locomotion

A semi-automated micro-electro-fluidic method to induce on-demand locomotion in Caenorhabditis elegans is described. This method is based on the neurophysiologic phenomenon of worms responding to mild electric fields (&#x201C;electrotaxis&#x201D;) inside microfluidic channels. Microfluidic electrotaxis serves as a rapid, sensitive, low-cost, and scalable technique to screen for factors affecting neuronal health.

基于微流体Electrotaxis的，为按需定量分析<em&gt;线虫</em&gt;&#39;LOCOMOTION的

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and ...

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson&#39;s disease. Acute activation of G&#945;i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of G&#945;i/o-linked receptors including D2 dopamine and &#956; opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gb&#947; signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384&#160;well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e.&#160;CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (&#62;20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening.

腺苷酸环化酶的药物引起的过敏性：含量简化和小型化的小分子和siRNA筛选应用

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

人体软骨组织制作采用三维喷墨打印技术

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Double Emulsion Generation Using a Polydimethylsiloxane (PDMS) Co-axial Flow Focus Device

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper presents a polydimethylsiloxane (PDMS) microfluidic device capable of generating water/oil/water double emulsions using a coaxial flow focusing geometry that can be fabricated entirely using soft lithography. Similar to emulsion devices using glass capillaries, double emulsions can be formed in channels with uniform wettability and with dimensions much smaller than the channel sizes. Three dimensional flow focusing geometry is achieved by casting a pair of PDMS slabs using two layer soft lithography, then mating the slabs together in a clamshell configuration. Complementary locking features molded into the PDMS slabs enable the accurate registration of features on each of the slab surfaces. Device testing demonstrates formation of double emulsions from 14 &#181;m to 50 &#181;m in diameter while using large channels that are robust against fouling and clogging.

Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

Microfluidic double emulsions generation typically involves devices with patterned wettability or custom-fabricated glass components. Here we describe the fabrication and testing of an all polydimethylsiloxane (PDMS) double emulsion generator that does not require surface treatment or complicated fabrication processes, and is capable of producing double emulsions down to 14 &#181;m.

复乳代使用聚二甲基硅氧烷（PDMS）同轴流量聚焦设备

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

使用多色荧光检测液滴微流控光纤

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

An Efficient and Reproducible Protocol for Distraction Osteogenesis in a Rat Model Leading to a Functional Regenerated Femur

This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) is a surgical technique leading to bone regeneration after an osteotomy. The osteotomized extremities are moved away from each other by gradual distraction to reach the desired elongation. This procedure is widely used in humans for lower and upper limb lengthening, treatment after a bone nonunion, or the regeneration of a bone defect following surgery for bone tumor excision, as well as in maxillofacial reconstruction. Only a few studies clearly demonstrate the efficiency of their protocol in obtaining a functional regenerated bone, i.e., bone that will support physiological weight-bearing without fracture after removal of the external fixator. Moreover, protocols for DO vary and reproducibility is limited by lack of information, making comparison between studies difficult. The aim of this study was to develop a reproducible protocol comprising an appropriate external fixator design for rat limb lengthening, with a detailed surgical technique that permits physiological weight-bearing by the animal after removal of the external fixator.

This study describes a reproducible and detailed protocol using a newly developed external fixator for distraction osteogenesis (DO) in a femoral rat model which permits physiological weight-bearing by the animal after removal of the external fixator.

大鼠牵张成骨的高效可再生性协议

This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) ...

Dual Raster-Scanning Photoacoustic Small-Animal Imager for Vascular Visualization

Imaging of vascular networks on small animals has played an important role in basic biomedical research. Photoacoustic imaging technology has great potential for application in the imageology of small animals. The wide-field photoacoustic imaging of small animals can provide images with high spatiotemporal resolution, deep penetration, and multiple contrasts. Also, the real-time photoacoustic imaging system is desirable to observe the hemodynamic activities of small-animal vasculature, which can be used to research the dynamic monitoring of small-animal physiological features. Here, a dual-raster-scanning photoacoustic imager is presented, featuring a switchable double-mode imaging function. The wide-field imaging is driven by a two-dimensional motorized translation stage, while the real-time imaging is realized with galvanometers. By setting different parameters and imaging modes, in vivo visualization of small-animal vascular network can be performed. The real-time imaging can be used to observe pulse change and blood flow change of drug-induced, etc. The wide-field imaging can be used to track the growth change of tumor vasculature. These are easy to be adopted in various areas of basic biomedicine research.

A dual raster-scanning photoacoustic imager was designed, which integrated wide-field imaging and real-time imaging.

用于血管可视化的双栅格扫描光声小动物成像仪

Imaging of vascular networks on small animals has played an important role in basic biomedical research. Photoacoustic imaging technology has great ...

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy

Subconjunctival injection is an attractive route to administer ocular drugs due to easy trans-scleral access that bypasses anterior ocular barriers, such as the cornea and conjunctiva. While therapeutic effects and pharmacokinetics of the drugs upon subconjunctival injection have been described in some studies, very few assess the ocular distribution of drugs or drug delivery systems (DDS). The latter is critical for the optimization of intraocular DDS design and drug bioavailability to achieve the desired ocular localization and duration of action (e.g., acute versus. prolonged). This study establishes the use of fiberoptic confocal laser microendoscopy (CLM) to qualitatively study the ocular distribution of fluorescent liposomes in real-time in live mice after sub-conjunctival injection. Being designed for in vivo visual inspection of tissues at the microscopic level, this is also the first full description of the CLM imaging method to study spatio-temporal distribution of injectables in the eye after subconjunctival injection.

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy

We present a protocol for the use of fiberoptic confocal laser microendoscopy (CLM) to non-invasively study the spatio-temporal distribution of liposomes in the eye after subconjunctival injection.

使用光纤共聚焦激光微内窥镜检查的眼部药物输送系统的时空 体内 成像

Subconjunctival injection is an attractive route to administer ocular drugs due to easy trans-scleral access that bypasses anterior ocular barriers, such ...