这项工作是由美国国立卫生研究院授予1R21NS064412，NSF资助1052048和Jeffress纪念信托基金资助的J-908的支持。

1，样品制备注：在许多情况下灌注实验洗去细胞的显著部分，它可以成为一个令人沮丧的问题。虽然没有必要对所有的细胞系，涂层覆盖玻璃表面用聚-L-赖氨酸（添加0.01％的溶液1.0 ml/25厘米2的表面，孵育&gt; 5分钟，用细胞培养级水洗涤两次，干燥生物安全柜）提高细胞的粘附。同时，要注意使用的细胞系的生物安全水平（BSL）的，并按照批准地方环境健康和安全办公室的标准作业程序。在该实验中，转染的COS7细胞用于由于低内源性谷氨酰胺转运活性（参见图4）。 <ol><li>种子COS7细胞在〜在Dulbecco改良的Eagle培养基（DMEM）70％-80％汇合度+10％宇宙的小牛血清和100 U / ml青霉素/链霉素，无论是在无菌25毫米盖玻片放置在6孔培养菜或8孔玻璃底的幻灯片。在37℃，5％CO 2，100％湿度下24小时。 </li><li>更换生长培养基，以DMEM +10％宇宙的小牛血清（不含抗生素），根据制造商的方案。成长O / N在37℃，5％CO2，100％湿度。 </li><li>将每个质粒DNA的0.4毫克编码谷氨酰胺传感器（染pcDNA3.1，携带FLIPQTV3.0传感器在CMV启动子10）和的mCherry标记ASCT2 10至50ml无血清培养基（OPTI-MEM），轻轻混匀。 </li><li>加入1 ml转染试剂至50ml无血清培养基。在室温下孵育5分钟。 </li><li>混合含有DNA（步骤1.3）和转染试剂（步骤1.4）的两种溶液并孵育20分钟。 </li><li>轻轻地加在细胞上的顶端的转染试剂和由摆动腔轻轻混匀。孵育24小时后，在37℃，5％CO 2，100％湿度。 </li><li> 24小时后，更换培养基，以DMEM +10％宇宙小牛血清（CCS）+ 100单位青霉素/链霉素。 </li><li>细胞成像48-72小时后转染。 </li></ol> 2，灌注实验注意：对于在该实验中使用COS7细胞，灌注介质和腔室保持在室温和环境CO 2浓度。然而，如果正在使用的电池需要较高的温度和CO 2浓度控制为生存，加热的显微镜载物台和/或环境室应该被使用。 <ol><li>准备灌注缓冲自动对焦（见材料清单）。附加旋塞至50ml注射器，关闭旋塞然后填写与灌注液。打开水龙头一个短暂的时期，以填补与缓冲活塞的头部空间。 </li><li>切换的8通道，重力进料灌注系统与灌注铅笔，然后选择在选择功能选项“手动模式”。放置一个废物容器下的灌注铅笔。 </li><li>手动通过按下相应的数字激活的端口，用10 ml注射器含有水补油管，然后关闭该端口。一旦端口被装满了水，设置包含缓冲区自动对焦灌注系统的端口1-6的注射器，然后打开旋塞。再次打开的端口，以取代水与缓冲管。流速应为约0.8毫升/分钟。 </li><li>按取消一次灌注控制器返回到“选择功能”菜单。切换到“编辑程序”选项，然后在灌注协议表1所示。保存的灌注程序。 </li><li>取出来的细胞培养箱中。与灌注缓冲液洗两次，然后将细胞在舞台上。灌注系统连接到室内。如果一个开放室使用，另设泵，去除室内的灌注液。 </li><li>打开Slidebook软件。开始一个新的幻灯片。 注意：下面的过程是特定于Slidebook软件，但其他软件如MetaFluor和萨 - 要素也可用于此处所述的成像的类型。理论上实验可以使用允许时间 - 过程成像任何软件来执行，并且图像序列可以被分析后的实验使用软件如ImageJ的（http://rsbweb.nih.gov/ij/）或斐济（呻/ fiji.sc /斐济）。然而，软件，允许的受体/供体比实时监测是非常有用的。 </li><li>安装滑道到荧光显微镜阶段。发现细胞共表达的传感器和ASCT-mCherry的使用适当的过滤器集（请参阅材料清单）下的“聚焦”功能。通过点击“相机”选项卡和“收益”项下滑动滑动条调节增益。此外，从调节中性密度滤镜下拉菜单中的中性密度滤镜设置。注意：如果过滤器多维数据集不包括眼滤镜，不要用眼睛片，因为肖尔的T波长的激发光对眼睛有害。使用电脑屏幕找到细胞来代替。 </li><li>获得的细胞图像with donor ( mTFP1) ex donor(mTFP1) em , donor ( mTFP1) ex acceptor(venus) em and acceptor ( mTFP1) ex acceptor(venus) em m channels （过滤器在材料列表中指定）。单击“图像捕捉”，然后在采集窗口，指定将要使用的过滤器。点击“测试”按钮，通过在框中键入所需的曝光时间旁边的过滤器设置来调整曝光时间。注：所有三个通道需要被暴露于相同的长度。当调节摄像参数，取预期的FRET效率的变化和折叠增加考虑：例如，如果预期的FRET效率在实验过程中向上去2倍，摄像参数应调整从而使供体的前接受器烯在静息状态下的强度应&lt;可被仪器记录在最大值的50％，从而使信道不会成为在实验过程中达到饱和。从标记细胞信号应至少比背景区域高3倍。 </li><li>使用该软件区域工具绘制一个地区的利益。可替换地，软件功能来选择上述一定强度的像素都可以使用，然后转换成的区域。要执行此操作，请单击面膜 - 分部，然后移动滑动条来突出显示所需的区域。 </li><li>选择在拍摄类型框中的“时间流逝”，然后指定时间间隔（10秒在这个实验）和实验时间点。 </li><li>在一个区域画出一个区域，没有任何荧光细胞。这个区域是用于背景减法。用鼠标右键单击绘制的区域，然后将其设置为背景。注意：在理想情况下，细胞是不吨表达该传感器应作为背景区域占两者的自体荧光和照相机所检测到的背景荧光。如果没有这样的细胞可在视场中，至少在没有细胞的区域应该被用来解释背景荧光。 </li><li>选项​​ - 在“运行程序选择功能”选择所需的程序。切换到保存的程序（见步骤2.3），然后打的灌注控制器上的ENTER键。现在，这个程序被加载，并且击中任何键将开始灌注。 </li><li>确保灌注和成像实验开始的同时按一下在同一时间按下灌注控制器上的按键，在拍摄窗口中的“开始”按钮。 注：建议记录那里的解决方案进行了交流（这可以通过使用“票据”功能，采集选项卡中找到来完成）的时间点。 </li><li>执行相同的灌注协议USI纳克细胞表达，预计将是饱和的或者未结合的实验条件下的传感器。 注意：在这里，FLIPQTV3.0_1.5μ，这是在实验条件下饱和的，被用作对照， 图6这样的对照实验是必要的，以确认该FRET效率的变化是由于在基板上的实际变化，而 ​​不是。由于在其它参数，如细胞内pH的变化。 </li></ol> 3，后期实验分析<ol><li>检查如果在实验过程中发生样品漂移。绘制感兴趣区域（ROI）区域S在0时间点拍摄的图像上，然后移动在成像窗口顶部的滑动杆，并检查电池掉出的ROI在后来的实验中拍摄的图像。 </li><li>如果是的话，那么使用跟踪功能来校正漂移，首先，在细胞周围建立口罩通过选择面膜 - 分类，然后将它指定截止到HIG行hlight区域含有细胞。追踪区域单击面膜 - 粒子跟踪 - 基本粒子跟踪。 </li><li>必要时重新绘制的ROI和背景区域。 </li><li>导出的感兴趣区的平均强度比实验的过程。点击统计 - 出口 - 比/慢速拍摄数据。 </li><li>当底物浓度的FRET效率和量化是没有必要的，画出作为基材的动态代理的强度比（ 供体前接受器烯 ，/供体的前施主烯 ）的时间过程。以确定细胞内浓度，计算出的最大和最小的比率变化（ 即，在所述基板的饱和和不存在，分别），ΔR 最大 - ΔR 分钟 ，由Δratio（ΔR）与下面的等式的非线性回归： ΔR= [S] /（K D + [S]）×（ΔR <suB&gt;最大值- ΔR 分钟 ）+△ 分钟 其中[S]是底物浓度在胞质溶胶中。使用ΔR 最大 -传感器得到ΔR 最小值 ，饱和度（S）在给定的ΔR被计算为 S =（ΔR - ΔR 分钟 ）/（ΔR 最大 - ΔR 分钟 ） S可进一步被转化成使用以下等式的底物浓度[S]： S = [S] /（K D + [S]） 注： 接受器前受体EM信道的强度应同时绘制研究实验条件是否影响受体的直接荧光。 </li><li>当基线漂移由于光漂白观察，进行基线校正。要做到这一点，画出供体和受体随着时间的强度（ 图5A）。然后确定用于日的时间点ë基线，根据在灌注系统中的延迟和特定的细胞（ 图5B）的转运活性。 </li><li>计算多项式拟合最能说明基线。然后，归一化的原始强度从每个时间点与基线（ 图5C）。从标准化的供体和受体的强度计算强度比（ 捐赠前受体他们，/捐赠者捐赠者除外EM）。 </li></ol>

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没有什么可以透露。

成像实验的成功取决于几个关键因素。其中一个因素是传感器的使用，如上面所讨论的亲和力。在感兴趣的亚细胞区室中衬底的绝对浓度，但是，常常是未知的。因此，我们建议您尝试多种传感器与交错的亲和力，以找到一个所需的实验条件下效果最好。例如，在我们的情况下，我们转染的COS7细胞中谷氨酰胺传感器，1.5μ，100μ，2分，和8米（ 图3和数据未显示）。 <p class="jove_conten...

由于技术的显着进步，使考试的转录组和蛋白质组在细胞水平上的，它现在已经很清楚，生物化学和由此产生的通量代谢物和离子是高度细胞类型特异性。例如，在哺乳动物的肝脏，顺序谷氨酰胺降解和合成是同时进行由门静脉周围的细胞和静脉周围的细胞分别，同时消耗过量的氨在后者的1-3喂养铵到尿素循环在前者的细胞类型。在某些情况下，显著生化异质性被检测，即使在一个单一的“小区类型”4，5。除了 ​​这样的空间特异性，代谢物和离子的细胞水平是高度动态的（ 例如，信号分子，如Ca 2 +，环 ​​核苷酸）。代谢物和离子的时空模式往往能起到关键作用的信号转导。 Monitoring代谢物和离子的细胞动力学，但是，提出了独特的挑战。在许多情况下，其浓度的变化是快速和短暂的，通过信号转导分子如Ca 2 +，内〜20毫秒的树突棘6，该衰变的情况举例说明。此外，内部和之间的细胞的生化途径的分隔使得难以用萃取和柱色谱/质谱技术定量代谢物和离子的动力学。 遗传编码的传感器，用于生物分子现在广泛由于高时空分辨率，允许实验者研究短命和/或隔离的分子动力学使用（7评论，8）。这些基因编码的传感器大致可以分为两大类;基于强度的传感器和ratiometic传感器。强度为基础的传感器通常由一个结合结构域和一个感orescent蛋白（FPS），以及溶质结合的结合结构域改变的荧光强度。 Ratiometic传感器，在另一方面，往往采取的促进共振能量转移（FRET）2的FP充当FRET对的优势。这些传感器包括一个结合结构域和两个FP的，与溶质结合诱导在2的FP之间的FRET效率的变化。大量的传感器，用于生物学上重要的代谢物和离子已被开发，在过去十年中8,9。 其中由该基因编码的传感器所提供的令人兴奋的可能性是他们在膜转运过程的高分辨率分析，这在以前是不容易察觉在细胞水平上的使用。遗传编码的传感器便利的运输机制，如底物特异性和pH依赖性10，11中的分析。此外，在同遗传水库组合OURCES如RNA干扰的库构建的模式生物，它现在可以进行全基​​因组搜索使用基因编码的传感器新颖的运输过程。事实上，利用基因编码的传感器导致的以前未知转运多例12，13的发现。 最近，我们实验室已经开发出一系列基于FRET的传感器谷氨酰胺。我们已经证明，细胞谷氨酰胺水平可以使用这样的FRET谷氨酰胺传感器10被可视化。这些传感器由在C-末端glnH的（ 图1）一个FRET供体（mTFP1）插入到细菌谷氨酰胺结合蛋白glnH和FRET受体（金星）的。这些传感器中的FRET效率下降时的谷氨酰胺结合，导致受体/供体的强度比的降低。谷氨酰胺转运过程的精细调节是重要的生物过程，如neurotransmission 14,15和尿素循环的维持在肝脏1，16，17。 在这里，我们将展示分析运输活动与FRET传感器谷氨酰胺，使用宽场荧光显微镜下建立的方法。这里显示的实验的目的是检测在单个细胞中转运活动，并检查一个瞬时表达转运蛋白的底物特异性。

<table border="0" cellpadding="0" cellspacing="0" width="1129">
	<tbody>
		<tr>
			<td>Inverted fluorescent microscope</td>
			<td>Olympus</td>
			<td>IX81F-3-5</td>
			<td>An equivalent inverted fluorescent microscope from other suppliers would also appropriate.</td>
		</tr>
		<tr>
			<td>Exitation filter (mTFP1)</td>
			<td>Omega Optical</td>
			<td>3RD450-460</td>
			<td>band width 450-460 nm</td>
		</tr>
		<tr>
			<td>Exitation filter (Venus)</td>
			<td>Chroma</td>
			<td>ET500/20</td>
			<td>band width 490-510 nm</td>
		</tr>
		<tr>
			<td>Emission filter (mTFP1)</td>
			<td>Chroma</td>
			<td>HQ495/30m</td>
			<td>band width 475-505nm</td>
		</tr>
		<tr>
			<td>Emission filter (Venus)</td>
			<td>Chroma</td>
			<td>ET535/30m</td>
			<td>band width 520-550nm</td>
		</tr>
		<tr>
			<td>Dichroic mirror (FRET channels)</td>
			<td>Chroma</td>
			<td>470dcxr</td>
			<td>long pass, 470nm cut off</td>
		</tr>
		<tr>
			<td>Dichroic mirror (YFP channel)</td>
			<td>Chroma</td>
			<td>89002bs</td>
			<td>passes 445-490nm, 510-560nm, 590-680nm</td>
		</tr>
		<tr>
			<td>mCherry filter set</td>
			<td>Chroma</td>
			<td>49008</td>
			<td>excitation 540-580nm, long pass dichroic with 585nm cut off, emission filter 595-695nm</td>
		</tr>
		<tr>
			<td>Light source</td>
			<td>Olympus</td>
			<td>U-LH100L-3-5</td>
			<td>LED- or halogen light source that produces stable light intensity, mercury lamps are not recommended</td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>Qimaging</td>
			<td>Rolera-MGi EMCCD</td>
		</tr>
		<tr>
			<td>apochromatic fluorescence objective</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>perfusion system, ValveBank II</td>
			<td>AutoMate Scientific</td>
			<td>[01-08]</td>
			<td>Other perfusion systems that allow fast solution exchange would also work</td>
		</tr>
		<tr>
			<td>Laminar-flow chambers</td>
			<td>C&#38;L Instruments</td>
			<td>VC-MPC-TW</td>
			<td>Other larminar-flow chambers would also work</td>
		</tr>
		<tr>
			<td>Chambered slide</td>
			<td>Lab-Tek</td>
			<td>154534</td>
			<td>For open-chamber experiments</td>
		</tr>
		<tr>
			<td>perfusion pump</td>
			<td>Thermo Scientific</td>
			<td>74-046-12131</td>
			<td>For open-chamber experiments</td>
		</tr>
		<tr>
			<td>Software supporting ratiometric measurements</td>
			<td>Intellegenent Imaging Innovation</td>
			<td>Slidebook 5.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar flow biosafety cabinet</td>
			<td>ESCO</td>
			<td>LA-3A2</td>
			<td></td>
		</tr>
		<tr>
			<td>Isotemp CO2 Incubator</td>
			<td>Thermo Scientific</td>
			<td>13-255-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s MEM (DMEM)</td>
			<td>Hyclone</td>
			<td>SH30243.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Cosmic Calf Serum</td>
			<td>Hyclone</td>
			<td>SH3008703</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin/streptomycin</td>
			<td>Hyclone</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>Serum-free medium for transfection (OPTI-MEM I)</td>
			<td>Invitrogen</td>
			<td>31985</td>
			<td>Used with Lipofectamine 2000</td>
		</tr>
		<tr>
			<td>Poly-L-Lysine solution</td>
			<td>sigma</td>
			<td>P4707</td>
			<td></td>
		</tr>
		<tr>
			<td>25mm circular glass cover slips</td>
			<td>Thermo Scientific</td>
			<td>12-545-102</td>
			<td>in case VC-MPC-TW is used</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>11668027</td>
			<td>Other transfection reagents can also be used</td>
		</tr>
		<tr>
			<td>Solution A (Hank&#39;s buffer)</td>
			<td>-</td>
			<td>-</td>
			<td>9.7g HANK salt (Sigma H1387), 0.35g NaHCO3, 5.96g HEPES to 1L, pH adjusted to 7.35 with NaOH</td>
		</tr>
		<tr>
			<td>Solution B</td>
			<td></td>
			<td></td>
			<td>Solution A + 0.04mM Gln</td>
		</tr>
		<tr>
			<td>Solution C</td>
			<td></td>
			<td></td>
			<td>Solution A + 0.2mM Gln</td>
		</tr>
		<tr>
			<td>Solution D</td>
			<td></td>
			<td></td>
			<td>Solution A + 1mM Gln</td>
		</tr>
		<tr>
			<td>Solution E</td>
			<td></td>
			<td></td>
			<td>Solution A + 5mM Gln</td>
		</tr>
		<tr>
			<td>Solution F</td>
			<td></td>
			<td></td>
			<td>Solution A + 5mM Ala</td>
		</tr>
	</tbody>
</table>

glutamine flux imaging using genetically encoded sensors

遗传编码的传感器允许生物分子的亚细胞分辨率的实时监控。一个巨大的品种，例如传感器，生物分子在过去15年中面世，其中一些成为了经常被用在许多实验室不可缺少的工具。 其中一个基因编码传感器的令人兴奋的应用之一是在调查细胞转运过程中使用这些传感器。转运如动力学和底物特异性的属性可以在细胞水平进行调查，为运输活动的细胞类型特异性分析的可能性。在这篇文章中，我们将演示如何转运动力学可以用基因编码的谷氨酰胺传感器为例进行观察。实验设计，实验设置的技术细节，和考虑后的实验分析将进行讨论。

典型时程实验示于图2，在这些实验中，FRET谷氨酰胺传感器为8毫米（FLIPQTV3.0_8m， 图2A和2B）和100μM（FlipQTV3.0_100μC和D）的亲和性被共表达以强制性氨基酸器ASCT2 18 COS7细胞10。谷氨酰胺流入被检测作为给体（mTFP1）和受体（金星）（ 图2A和2C）之间的荧光强度比的变化。谷氨酰胺在另一衬底（丙氨酸）的存在下的流出也?...

Watch this Scientific Journal Video about Glutamine Flux Imaging Using Genetically Encoded Sensors at JoVE.com

Glutamine Flux Imaging Using Genetically Encoded Sensors

本文将展示如何利用FRET监测谷氨酰胺动态活细胞。遗传编码的传感器允许生物分子的亚细胞分辨率的实时监控。实验设计，实验设置的技术细节，并考虑后期的实验分析将用于基因编码的谷氨酰胺的传感器进行讨论。

谷氨酰胺通量成像使用基因编码的传感器

Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. A tremendous variety of such sensors for ...

glutamine-flux-imaging-using-genetically-encoded-sensors

Research

JoVE Journal

Bioengineering

9.5K 次观看.  Virginia Tech. 本文将展示如何利用FRET监测谷氨酰胺动态活细胞。遗传编码的传感器允许生物分子的亚细胞分辨率的实时监控。实验设计，实验设置的技术细节，并考虑后期的实验分析将用于基因编码的谷氨酰胺的传感器进行讨论。 

视频: Glutamine Flux Imaging Using Genetically Encoded Sensors

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

使用电化学传感器的细菌检测和鉴定

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

科研

生物工程

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

微流体为基础的电化学生物芯片的无标记的DNA杂交分析

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

Flying Insect Detection and Classification with Inexpensive Sensors

An inexpensive, noninvasive system that could accurately classify flying insects would have important implications for entomological research, and allow for the development of many useful applications in vector and pest control for both medical and agricultural entomology. Given this, the last sixty years have seen many research efforts devoted to this task. To date, however, none of this research has had a lasting impact. In this work, we show that pseudo-acoustic optical sensors can produce superior data; that additional features, both intrinsic and extrinsic to the insect&#8217;s flight behavior, can be exploited to improve insect classification; that a Bayesian classification approach allows to efficiently learn classification models that are very robust to over-fitting, and a general classification framework allows to easily incorporate arbitrary number of features. We demonstrate the findings with large-scale experiments that dwarf all previous works combined, as measured by the number of insects and the number of species considered.

We proposed a system that uses inexpensive, noninvasive pseudo-acoustic optical sensors to automatically and accurately detect, count, and classify flying insects based on their flying sound.

飞行昆虫检测与分类用便宜的传感器

An inexpensive, noninvasive system that could accurately classify flying insects would have important implications for entomological research, and allow ...

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 &#181;m diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

基因转染向着文化微模式球体板细胞基因修饰的细胞移植

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic micro&#64258;uidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

使用多通道双电场微流控芯片肺癌细胞Electrotaxis研究

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem cell (iPS) emerged as a new method in regenerative medicine. Although this tissue regeneration is promising, contamination with unwanted cells during tissue cultures is a major concern. Moreover, there is a safety concern regarding tumorigenicity after transplantation. Therefore, there is an urgent need for eliminating specific cells without damaging other cells that need to be protected, especially in established tissue. Here, we present a method for specific cell elimination from a mixed 3D cell culture in vitro with near infrared photoimmunotherapy (NIR-PIT) without damaging non-targeted cells. This technique enables the elimination of specific cells from mixed cell cultures or tissues.

Eliminating specific cells without damaging other cells is extremely difficult, especially in established tissue, yet there is an urgent need for a cell elimination method in the tissue engineering field. Here, we present a method for specific cell elimination from a mixed 3D cell culture using near infrared photoimmunotherapy (NIR-PIT).

从混合三维培养使用近红外Photoimmunotherapy技术选择性细胞消除

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem ...

Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. Biosensors relying on the principle of FRET have enabled real-time visualization of subcellular signaling events in live cells with high temporal and spatial resolution. Here, we describe the application of a genetically encoded Bile Acid Sensor (BAS) that consists of two fluorophores fused to the farnesoid X receptor ligand binding domain (FXR-LBD), thereby forming a bile acid sensor that can be activated by a large number of bile acids species and other (synthetic) FXR ligands. This sensor can be targeted to different cellular compartments including the nucleus (NucleoBAS) and cytosol (CytoBAS) to measure bile acid concentrations locally. It allows rapid and simple quantitation of cellular bile acid influx, efflux and subcellular distribution of endogenous bile acids without the need for labeling with fluorescent tags or radionuclei. Furthermore, the BAS FRET sensors can be useful for monitoring FXR ligand binding. Finally, we show that this FRET biosensor can be combined with imaging of other spectrally distinct fluorophores. This allows for combined analysis of intracellular bile acid dynamics and i) localization and/or abundance of proteins of interest, or ii) intracellular signaling in a single cell.

We provide a detailed protocol to study bile acid dynamics in living cells using a genetically encoded BAS FRET sensor. This Bile Acid Sensor represents a unique tool to study (regulation of) bile acid transport and FXR activation in a wide range of cell types.

细胞内胆汁酸动态实时监控使用遗传编码的FRET为基础的胆汁酸传感器

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. ...

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds during repair, which gives rise to a lack of sufficient blood supply and causes necrosis of the new tissues. Rapid vascularization is a vital prerequisite for new tissue survival and integration with existing host tissue. The de novo generation of vasculature in scaffolds is one of the most important steps in making bone regeneration more efficient, allowing repairing tissue to grow into a scaffold. To tackle this problem, the genetic modification of a biomaterial scaffold is used to accelerate angiogenesis and osteogenesis. However, visualizing and tracking in vivo blood vessel formation in real-time and in three-dimensional (3D) scaffolds or new bone tissue is still an obstacle for bone tissue engineering. Multiphoton microscopy (MPM) is a novel bio-imaging modality that can acquire volumetric data from biological structures in a high-resolution and minimally-invasive manner. The objective of this study was to visualize angiogenesis with multiphoton microscopy in vivo in a genetically modified 3D-PLGA/nHAp scaffold for calvarial critical bone defect repair. PLGA/nHAp scaffolds were functionalized for the sustained delivery of a growth factor pdgf-b gene carrying lentiviral vectors (LV-pdgfb) in order to facilitate angiogenesis and to enhance bone regeneration. In a scaffold-implanted calvarial critical bone defect mouse model, the blood vessel areas (BVAs) in PHp scaffolds were significantly higher than in PH scaffolds. Additionally, the expression of pdgf-b and angiogenesis-related genes, vWF and VEGFR2, increased correspondingly. MicroCT analysis indicated that the new bone formation in the PHp group dramatically improved compared to the other groups. To our knowledge, this is the first time multiphoton microscopy was used in bone tissue-engineering to investigate angiogenesis in a 3D bio-degradable scaffold in vivo and in real-time.

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

Here, we present a protocol to visualize blood vessel formation in vivo and in real-time in 3D scaffolds by multiphoton microscopy. Angiogenesis in genetically modified scaffolds was studied in a murine calvarial critical bone defect model. More new blood vessels were detected in the treatment group than in controls.

多显微镜下的血管生成术在基因修饰的 3 d-PLGA/nHAp 支架上进行颅骨临界骨缺损修复

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds ...

Production of Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex

The pronuclear (PN) injection technique was first established in mice to introduce foreign genetic materials into the pronuclei of one-cell stage embryos. The introduced genetic material may integrate into the embryonic genome and generate transgenic animals with foreign genetic information following transfer of the injected embryos to foster mothers. Following the success in mice, PN injection has been applied successfully in many other animal species. Recently, PN injection has been successfully employed to introduce reagents with gene-modifying activities, such as the CRISPR/Cas9 system, to achieve site-specific genetic modifications in several laboratory and farm animal species. In addition to mastering the special set of microinjection skills to produce genetically modified animals by PN injection, researchers must understand the reproduction physiology and behavior of the target species, because each species presents unique challenges. For example, golden Syrian hamster embryos have unique handling requirements in vitro such that PN injection techniques were not possible in this species until recent breakthroughs by our group. With our species-modified PN injection protocol, we have succeeded in producing several gene knockout (KO) and knockin (KI) hamsters, which have been used successfully to model human diseases. Here we describe the PN injection procedure for delivering the CRISPR/Cas9 complex to the zygotes of the hamster, the embryo handling conditions, embryo transfer procedures, and husbandry required to produce genetically modified hamsters.

Pronuclear (PN) injection of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein-9 nuclease (CRISPR/Cas9) system is a highly efficient method for producing genetically engineered golden Syrian hamsters. Herein, we describe the detailed PN injection protocol for the production of gene knockout hamsters with the CRISPR/Cas9 system.

CRISPR/Cas9 复合原注射液生产基因工程金黄叙利亚仓鼠

The pronuclear (PN) injection technique was first established in mice to introduce foreign genetic materials into the pronuclei of one-cell stage embryos. ...

Targeting Neuronal Fiber Tracts for Deep Brain Stimulation Therapy Using Interactive, Patient-Specific Models

Deep brain stimulation (DBS), which involves insertion of an electrode to deliver stimulation to a localized brain region, is an established therapy for movement disorders and is being applied to a growing number of disorders. Computational modeling has been successfully used to predict the clinical effects of DBS; however, there is a need for novel modeling techniques to keep pace with the growing complexity of DBS devices. These models also need to generate predictions quickly and accurately. The goal of this project is to develop an image processing pipeline to incorporate structural magnetic resonance imaging (MRI) and diffusion weighted imaging (DWI) into an interactive, patient specific model to simulate the effects of DBS. A virtual DBS lead can be placed inside of the patient model, along with active contacts and stimulation settings, where changes in lead position or orientation generate a new finite element mesh and solution of the bioelectric field problem in near real-time, a timespan of approximately 10 seconds. This system also enables the simulation of multiple leads in close proximity to allow for current steering by varying anodes and cathodes on different leads. The techniques presented in this paper reduce the burden of generating and using computational models while providing meaningful feedback about the effects of electrode position, electrode design, and stimulation configurations to researchers or clinicians who may not be modeling experts.

The goal of this project is to develop an interactive, patient-specific modeling pipeline to simulate the effects of deep brain stimulation in near real-time and provide meaningful feedback as to how these devices influence neural activity in the brain.

应用交互式、患者特异性模型对神经纤维束进行深部脑刺激治疗的靶向性

Deep brain stimulation (DBS), which involves insertion of an electrode to deliver stimulation to a localized brain region, is an established therapy for ...