这项工作是由来自法新社国立补助倒拉RECHERCHE，PIRIbio程序Dynabiofilm和法国国家科学研究中心的跨学科风险项目支持的一部分。我们感谢菲利普托门对他的批判的手稿和Christophe Beloin阅读提供了E。大肠杆菌菌株在这项工作中使用。

1，细菌培养及悬浮法制备<ol><li>从溶原性肉汤（LB）琼脂平板上挑新鲜生长的菌落，接种在含有100μ微克/毫升氨苄青霉素和7.5μ克/毫升四环素的LB液体培养基中5毫升孵育它为5至6小时，在37℃上用颤抖的平台。 </li><li>然后，在5毫升的最小培养基（M63B1），补充了0.4％葡萄糖和相同浓度的抗生素添加的细菌培养物100μ升。这孵育新鲜稀释的培养过夜，于37℃用颤抖的平台上。 </li><li> 16小时培养后，μL过夜培养增加100至5ml M63B1，0.4％葡萄糖。保持所述管在37℃至摇动平台，直到0.5外径为止。该悬浮液，然后准备好用于注射到对生物膜形成的实验通道。 </li></ol> 2，磁粉制备<ol><li>取10μl的磁性粒子 -2.8μ米直径-从股票和它们洗干净3倍于190微升基本培养基用磁性样品架的援助。 </li><li>调整颗粒浓度为5×10 6珠/毫升。通常为50微升的洗涤珠溶液的混合与另外的950μ升M63B1用0.4％葡萄糖。 </li></ol> 3，渠道准备和生物膜生长<ol><li>通道安装<ol><li>切两方（800μ米边长），硼硅玻璃毛细管长10厘米，以获得两个8厘米长的块。 </li><li>胶水2的毛细管片上的两个载玻片-切成两半第一-相距2厘米与1厘米悬任一端上， 如图1使用速效氰基丙烯酸酯胶（所谓的超级胶水）。 </li><li>高压处理整个设置和需要进行进一步的通道连接所需的管材。 </li><li>聚集下的所有无菌材料在层流罩是:i）安装在通道和陷阱1，ⅱ）管和连接器，iii）该2气泡陷阱 - 泡沫过滤器通常用于保护儿童滴哺（陷阱1）和自制的气泡阱作为大直径的4厘米长的管子（陷阱2），IV）夹具，V）30毫升注射器装满M63B1，0.4％葡萄糖，及vi）废液瓶。 </li><li>连接整个安装与鲁尔锁按以下顺序连接到电脑或或路口:50毫升M63B1注射器，注射泵，儿科泡沫过滤器，自制的泡沫陷阱，毛细管（ 图1，图B），管到废液瓶控制。然后填入安装用无菌M63B1，0.4％葡萄糖，以约10毫升/小时，比实验速率更高的速率打开注射泵。仔细地跟踪和消除电路中的所有气泡。 </li><li>通过该系统为10-15分钟流动的介质;同时将1毫升细菌悬液在OD 0.5从第1.3用1毫升2.2节准备的冲珠液。 </li><li>附加（但不要关闭）夹在油管在两个位置:前后毛细血管。切换流过。 </li><li>引入细菌珠混合物进入毛细管用1毫升注射器自制泡沫陷阱后，小心地托起管端，以防止空气进入。重新连接油管，然后关闭夹子。 </li><li>重复同样的步骤进行的第二毛细管和检查所有的管气泡。 </li><li>该装置转移到显微镜，并允许它静置15-20分钟，以使细菌沉降并附着在毛细管的表面上。安装在毛细管上的显微镜载物台用在稍高的水平的废物容器中。放置在显微镜旁边的柜台上方的注射泵。毛细管比毛细管平面稍高捕捉气泡前提高了泡沫陷阱。 </li></ol></li><li>生物膜格罗问心无愧<ol><li>通常在24或48小时在这些实验 - 调整在注射器泵的流速，并开始流动，生物膜现在将在规定期间开发的毛细管表面。 </li><li>专注于毛细管底平面，并开始样品图像的时间间隔录制 - 2张图片通常是采集频率/分钟，会充分报告生物膜生长。此视频监视器生物膜生长后期控制（见中提取视频1，2和3）。 </li></ol></li></ol> 4，磁性镊子安装<ol><li>拧上手动控制XYZ显微操作的磁性镊子和螺丝显微镜舞台上的显微操作来调整镊子的位置相对于毛细管。将镊子如在图2，以确保适当的磁场梯度是在观察区产生。 </li><li>连接镊子吨Ø通过功率放大器函数发生器产生的时间40秒内由24秒零信号和16秒4的直流电用20秒的信号同步后发送到明视场光闸提供的序列触发事件，在图3中 。 注:这两个操作可以在毛细管的安装和测量开始之间的任何时间来实现。见实验概述草图如图4所示。 </li></ol> 5，蠕变曲线收购<ol><li>使用显微镜载物台的XY移动控制，使左侧的磁极，并在相同的观察视野的毛细血管的左手边缘的边缘。取分析参照的原点在x-和通过毛细管的边缘限定的y坐标轴的交点和磁极片的边缘上，分 ​​别（ 见图2）。 </li><li>用细焦点c调整毛细管的垂直位置显微镜的位置ONTROL旋钮。通常情况下，第一检测平面位于介于4至7μ米的毛细管底部的上方。 视频1对应于xy字段具有其顶部的左上角处的空间参照的原点。 </li><li>触发通过对电流发生器，并在同一时间开关在4.2节和图3描述的事件的40秒的顺序，手动触发录像1的图像采集序列。 </li><li>将毛细管的邻居右场由显微镜载物台的一个250μ米翻译到左边，并作为在第5.3节，以产生视频切片2的2等以获得所需的视频。为250×250μ 平方公尺通常为3至4场是沿改变平面并且重复相同的操作，对于新平面之前的x轴收集。 </li></ol> 6。力校准<ol><li>通过混合39.8克甘油与190μ升双蒸水和10微升的磁性颗粒在2×10 9个/毫升准备甘油溶液，并填写实验频道与此混合物，并把它放在显微镜载物台如对于生物膜样品。 </li><li>安装磁性镊子如第4所示后，将在第5.4节所指示的磁力，使时间推移图像提取每个单粒子速度（v）和毛细管的位置，以获得校准文件。此文件应包含所施加的力作为其在毛细管分析的区域位置的函数，根据Stokes定律，F =6πRηv（R:颗粒半径）。 </li></ol> 7。分析<ol><li>使用"粒子跟踪器"的软件来获取文本文件，用于收购如第5所示的图像的所有堆在每一帧中颗粒归仓。使用设置克图像堆栈采集频率，计算颗粒的位移与时间的函数（ 例如， 图5和视频4）。 </li><li> :根据符合公式使用强制校准文件，转换成位移曲线，以符合曲线（作为时间的函数的材料完全符合- -嘉（T）） <img alt="" fo:content-width="1in" src="/files/ftp_upload/50857/50857eq1.jpg" width="106" /> 这使材料应变之间的关系和所施加的应力为半径R的嵌入在一种不可压缩的，均匀的粘弹性介质如先前由Schnurr和同事19建立的探针粒子。 </li><li>调整蠕变柔量曲线的粘弹性材料的一般Burgers模型，并推导出粘弹性参数，J 0，J 1，η0，η1为每个粒子（ <strong&gt;图6）根据本伯格斯式: <img alt="" fo:content-width="2in" src="/files/ftp_upload/50857/50857eq2.jpg" width="169" /> 注意:此现象学分析先前已经用于各种各样的材料，包括生物材料，例如生物膜来解释宏观流变数据20-22。 </li></ol>

<ol>
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我们什么都没有透露。

此磁性粒子播种和拉试验中的一个生长的生物膜在其原始状态下的粘弹性参数的原位三维测绘启用。这种方法揭示了大肠杆菌的力学性能不均匀性这里大肠杆菌生物膜的生长，给了线索指出生物膜元器件配套生物膜的物理性能，强烈暗示了细胞外基质的基本内涵和更精确的交联度。 在细菌生物膜的机械性能模式的识别是建立这些复杂的材料进行全面的代表性至关重要。这些发现开辟澄清联机械纹和生物异质性，如基因表达microniches，这应该有助于澄清的驱动力支撑生物膜发展的因果关系的方式。 到现在为止，生物膜机器人的问题nical物业已解决主要从宏观的角度，往往是从最初的网站8,9,12,13，它代表信息丢失的重要危险刮生物膜。我们的非侵入性的方法带来了首次在其原始环境中的生物膜3D机械配置的原位表征。 该方法还具有驻留在胶体探针的磁性遥控致动的特性的限制。事实上，它显示由极粒子的距离，极材料性能和线圈性能给出访问势力的固有范围。的设置当前配置启用刚性值高达200帕，这是足以让这里研究的生物膜探测。尽管如此，我们的设置进一步的工程 - 涉及，例如，电磁铁冷却 - 应以2〜3倍的能力和时间的限制转移。 WË目前正在就有关在生物膜调查报告的链接通过探头的被动位移在更大的时间尺度局部粘弹性参数的关系的物理和动力学性质。此外，我们正在探索关于它的机械配置针对生物膜的不同组成部分的各种化学物质的影响。 这种新的方法揭示了细菌生物膜内部机制的细节，有助于更好地理解这些活结构，关键但从工程师的角度对生物膜的控制目的，而且从基本面看，以澄清建筑性质和具体之间的关系生物学这些结构。

细菌生物膜与生物或人工表面1-3相关的细菌群落。他们被加上生产多糖丰富的细胞外基质，保护和稳定的大厦4,5的附着生长机制形成。这些生物膜是细胞不会粘到表面的简单被动的组合，但有组织的和动态的复杂的生物系统。当细菌从浮游切换到生物膜的生活方式，改变基因表达和细胞生理学观察以及增加对抗菌性和宿主免疫防御是在许多长期存在的慢性感染6的由来。然而，这些生活结构的控制发展还为工业和环境应用，如生物修复的危险废物的场所，生物过滤工业用水或形成生物屏障，以保护土壤和地下水康泰明机会通报BULLETIN。 而具体到生活的方式生物膜分子特征越来越描述，带动社区发展和持久性的机制尚不清楚。使用上，使用扫描电化学或荧光显微镜微尺度测量的最新进展，这些活组织已表明具有相当大的结构，化学和生物异质7。然而，到现在为止，生物膜力学一直主要研究宏观。例如，观察生物膜拖缆的变形，由于变化的流体流速8,9，生物膜件单轴压缩解除从琼脂培养基或生长在盖玻片10,11，生物膜从环境中收集的剪切，然后转移到一个并行板流变仪12,13，采用了玻璃珠和涂有细菌生物膜附着于AFM悬臂14或专用的磁墨字符识别的原子力显微镜ocantilever测定方法分离的生物膜片段15,16的拉伸强度在10过去几年已经实现，提供对材料17的粘弹性性质的有用信息。然而，这很可能是在原位生物膜的机械性能信息时，该材料是从它的天然环境中，这是经常在这些方法的情况下除去丢失。另外，生物膜的治疗，作为均质材料射门对社区内的物理性能的可能的异质性的信息。因此，在生物膜形成的结构力学和生物特性，如基因表达的图案化或化学梯度的确切含义也很难被确认。要对生物膜的物理性能的微观描述的进步，新的专用工具是必需的。 本文详细构思，以达到原始的方法测量的原位局部机械参数，而不会干扰生物膜和有利的微尺度材料性质的空间分布绘图，然后在机械异质性。该实验的原理建立在一个生长生物膜与磁性体微粒随后通过远程装入利用磁镊子的成熟生物膜中的掺杂。在显微镜下成像控制磁力应用质点位移使当地的黏弹性参数导出，每个粒子报告自己的本地环境。从这些数据中，生物膜的三维机械更新可以得出，揭示空间和环境条件的依赖性。整个实验将在这里展示在大肠杆菌被大肠杆菌生物膜通过基因工程菌株携带去阻遏的F-质粒一样做。在最近的一篇文章18所详述的结果提供了完整的生物膜力学的内部的独到眼光。

<table border="0" cellpadding="0" cellspacing="0" width="1258">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td colspan="5" height="21" style="height:21px;width:1257px;">Table 1: Reagents and cells</td>
		</tr>
		<tr height="18">
			<td height="20" rowspan="2" style="height:22px;width:216px;">Magnetic particles</td>
			<td rowspan="2" style="width:323px;">Life technologies</td>
			<td rowspan="2" style="width:224px;">14307D</td>
			<td colspan="2" rowspan="2" style="width:496px;">Micrometric magnetic particle, 2.8 &#181;m diameter</td>
		</tr>
		<tr height="2">
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">Ampicillin (Antibiotic)</td>
			<td style="width:323px;">Sigma-Aldrich</td>
			<td style="width:224px;">A9518</td>
			<td colspan="2" style="width:496px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tetracycline (Antibiotic)</td>
			<td style="width:323px;">Sigma-Aldrich</td>
			<td style="width:224px;">87128</td>
			<td colspan="2" style="width:496px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bacterial strain MG1655gfpF</td>
			<td style="width:323px;">UGB, Institut Pasteur, France</td>
			<td style="width:224px;"></td>
			<td colspan="2" style="width:496px;">produces F pili at its surface, resistant to Ampicilllin and tetracycline</td>
		</tr>
		<tr height="21">
			<td colspan="5" height="21" style="height:21px;width:1257px;">Table 2:&#160; Capillaries and tubing</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:216px;">Filters for pediatric perfusion</td>
			<td style="width:323px;">Prodimed-Plastimed</td>
			<td style="width:224px;">6932002</td>
			<td colspan="2" style="width:496px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Hollow Square Capillaries</td>
			<td style="width:323px;">Composite Metal Scientific</td>
			<td style="width:224px;">8280-100</td>
			<td colspan="2" style="width:496px;">Manufactured in Borosilicate glass. Square 0.8mm x0.8mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tubing silicone peroxyde</td>
			<td style="width:323px;">VWR international</td>
			<td style="width:224px;">228-0512</td>
			<td colspan="2" style="width:496px;">Diameter 1mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tubing silicone peroxyde</td>
			<td style="width:323px;">VWR international</td>
			<td style="width:224px;">228-0700</td>
			<td colspan="2" style="width:496px;">Diameter 3mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Table 3: Biofilm growth</td>
			<td style="width:323px;"></td>
			<td style="width:224px;"></td>
			<td style="width:495px;"></td>
			<td style="width:1px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lysogeny Broth (LB) solution</td>
			<td style="width:323px;">Amresco-VWR</td>
			<td style="width:224px;">J106-10PK</td>
			<td colspan="2" style="width:496px;">standard medium used to grow bacteria</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">M63B1 solution</td>
			<td style="width:323px;">Home-made</td>
			<td style="width:224px;"></td>
			<td colspan="2" style="width:496px;">Standard minimum&#160; medium used to grow bacteria</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glucose</td>
			<td style="width:323px;">Sigma-Aldrich</td>
			<td style="width:224px;">G8270</td>
			<td colspan="2" style="width:496px;">Used to make M63B1 medium with 0.4% glucose</td>
		</tr>
		<tr height="21">
			<td colspan="5" height="21" style="height:21px;width:1257px;">Table 4: Electronics</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Camera EMCCD&#160;&#160;</td>
			<td style="width:323px;">Hamamatsu</td>
			<td style="width:224px;">C9100-02</td>
			<td colspan="2" style="width:496px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">Heater controller</td>
			<td style="width:323px;">World precision instruments</td>
			<td style="width:224px;">300354</td>
			<td colspan="2" style="width:496px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Function generator</td>
			<td style="width:323px;">Agilent technologies</td>
			<td style="width:224px;">33210A</td>
			<td colspan="2" style="width:496px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Power amplifier</td>
			<td style="width:323px;">Home-made</td>
			<td style="width:224px;"></td>
			<td colspan="2" style="width:496px;">It gives a current signal with amplitudes up to 4 A.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Syringe pumps</td>
			<td style="width:323px;">Kd Scientific</td>
			<td style="width:224px;">KDS-220</td>
			<td colspan="2" style="width:496px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Shutter</td>
			<td style="width:323px;">Vincent Associates</td>
			<td style="width:224px;">Uniblitz T132</td>
			<td colspan="2" style="width:496px;"></td>
		</tr>
		<tr height="125">
			<td height="125" style="height:125px;width:216px;">Magnetic tweezers</td>
			<td style="width:323px;">Home-made</td>
			<td style="width:224px;"></td>
			<td colspan="2" style="width:496px;">Two electromagnetic poles, each made of a copper coil with 2,120 turns of 0.56 mm in diameter copper wire and soft magnetic alloy cores (Supra50-Arcelor Mittal, France) square shaped according to the blueprint shown in Fig. 10. The two cores are mounted north pole facing south pole, in order to generate a magnetic force in one direction along the length of the capillary. See coil wiring details in Figure 11.</td>
		</tr>
		<tr height="21">
			<td colspan="5" height="21" style="height:21px;width:1257px;">Table 5: Optics</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Inverted microscope&#160;</td>
			<td style="width:323px;">Nikon</td>
			<td style="width:224px;">TE-300</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">S Fluor x40 Objective (NA 0.9, WD0.3)</td>
			<td>Nikon</td>
			<td style="width:224px;"></td>
			<td>This a long working distance ojective enabling observation of the biofilm in the depth</td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Epifluorescence filters: 1) for green fluorescence: Exc 480/20 nm; DM 495; Em 510/20&#160; 2) for Red fluorescence: Exc 540/25 nm; DM 565; Em 605/55</td>
			<td style="width:323px;">Chroma</td>
			<td style="width:224px;">1)#49020 2)#31002</td>
			<td>Particle displacement upon force application is recorded using the red fluoresecnce filter block.</td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="5" height="21" style="height:21px;width:1257px;">Table 6: Image analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ImageJ</td>
			<td style="width:323px;">NIH - particle tracker plugin</td>
			<td style="width:224px;"></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

remote magnetic actuation of micrometric probes for in situ 3d mapping of bacterial biofilm physical properties

细菌粘附和生长在接口导致的立体异构结构的所谓的生物膜的形成。细胞蜗居在这些结构是由胞外聚合物网络介导的物理相互作用结合在一起。细菌生物膜的影响许多人的活动及其性质的理解是至关重要的一个更好的控制其发展 - 维护或消灭 - 这取决于它们的不利或有利的结果。本文介绍了一种新的方法，旨在原位测量已被生物膜的本地物理性质，到现在为止，只检查从宏观和均质材料的观点。这里描述的实验中涉及引入磁性颗粒进入生物膜生长的种子，可以在不干扰生物膜的结构特性被远程致动本地探针。专用的磁性镊子是德韦大步施加一个力定义嵌入式生物膜每个粒子。安装被安装在显微镜的阶段，以使粒子拉期间的时间推移的图像的记录。粒子轨迹然后从拉序列提取与本地粘弹性参数从每个粒子位移曲线得出，由此提供的参数的三维空间分布。获得见解生物膜的机械配置是必不可少但从工程师的角度对生物膜的控制目的，而且从基本面看，澄清的建筑特性和这些结构的具体生物学之间的关系。

一个典型的分析将提供粘弹性参数的空间分布上的一个活生生的生物膜微米尺度不影响到其原来的安排。典型的结果示于图7，其中J 0的值-弹性柔-被给定为z轴沿y轴沿生物膜的横向尺寸的深度和的函数。每个点对应的蠕变曲线分析提供了为J 0值珠子。数据显示，当地合规沿着生物膜的深度超过幅度近三个数量级，但也是强大的横向不均匀性变化发生在所有生物膜的高度。 该数据也给访问哪些在这里展出了广泛的和不对称的形状（ 图8），提供了强有力的迹象表明合规值的分布是生物膜的力学性能进行了支持高度交联的聚合物凝胶。实际上，类似的行为已经预先证明对浓缩和高度交联的肌动蛋白的凝胶23。 此外，这些中的不同的环境条件下进行的，例如较低的流速生物膜局部特性原位测量启用以证明在生物膜的内部组织这样的变化的影响。在图9中 ，以0.1毫升/小时生长的生物膜的顺应性值显示出仍然高度异质机械更新，但更深一层生物膜的任何更高的刚性相比，以1毫升/小时的生长生物膜。这些结果证明的外部条件对生物膜组织的显著影响。 <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/50857/50857fig1highres.jpg" src="/files/ftp_upload/50857/50857fig1.jpg" /> <st<html>荣&gt;图1。毛细管安装草图。 <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/50857/50857fig2highres.jpg" src="/files/ftp_upload/50857/50857fig2.jpg" /> 图2。磁镊子相对于显微镜舞台上和空间参考的定义中的毛细管的定位的顶视图，表示用测量区域中的毛细管的位置。 <img alt="图3" fo:content-width="5in" fo:src="/files/ftp_upload/50857/50857fig3highres.jpg" src="/files/ftp_upload/50857/50857fig3.jpg" /> 图3。同步随着时间的40秒周期的信号。 857/50857fig4.jpg"/&gt; 图4。实验概述草图。 <img alt="图5" fo:content-width="5in" fo:src="/files/ftp_upload/50857/50857fig5highres.jpg" src="/files/ftp_upload/50857/50857fig5.jpg" /> 图5。从粒子轨迹分析萃取物。图A显示在左边的典型颗粒的图像在刚刚磁力应用（时间23.79秒的40秒期间）前的初始位置，并在右侧的粒子的位置时间30秒，连同使用ImageJ粒子跟踪图B显示了从这些数据得出的位移曲线的情节中找到轨迹的显示;在这里我们展示一个轨迹提取物在开始时23.79秒最多时间t = 31.29秒的40秒的时期。 <img alt="图6" fo:content-width="5in" fo:src="/ files/ftp_upload/50857/50857fig6highres.jpg"SRC ="/ files/ftp_upload/50857/50857fig6.jpg"/&gt; 汉堡的唯象模型的基础上，如图6所示。粘弹性参数推导（A）汉堡力学模型由弹性虎克弹簧（j 0）和串联一个麦克斯韦元件（弹簧（牛顿粘壶（η0）的组合□1）和缓冲器（η1）并联）。 （ 二）蓝色蠕变实验跟踪与曲线（绿色）调整到Burgers方程。 <img alt="图7" fo:content-width="5in" fo:src="/files/ftp_upload/50857/50857fig7highres.jpg" src="/files/ftp_upload/50857/50857fig7.jpg" /> 图7。弹性柔度的下1毫升生长24小时，在37℃生物膜的空间分布/小时养分流动 。报告的每个粒子符合价值观<html>这里被表示为使用冷色到暖色自定义颜色映射投影到（YZ）平面。报告由位于生物膜的底层颗粒遵守值都低于0.2米2 / N，而在高于1米2 / N。上部15μ米层报告值发现粒子<img alt="图8" fo:content-width="5in" fo:src="/files/ftp_upload/50857/50857fig8highres.jpg" src="/files/ftp_upload/50857/50857fig8.jpg" /> 图8中的生物膜（A）和甘油（B）中得到的粘弹性参数，均匀的粘稠液体的归一化分布。生物膜表现出非对称分布范围广（3.6偏度和归一化方差2.4）表示的材料的异质性。相比之下，对称分布窄表征均匀介质中的甘油（0.23和归一化方差0.03偏斜度）获得。 ontent"FO:保持together.within页="总是"&gt; <img alt="图9" fo:content-width="5in" src="/files/ftp_upload/50857/50857fig9highres.jpg" width="500" /> 图9。弹性柔顺的在0.1毫升/小时的营养素流更低的流动生长24小时后，在37℃的生物膜的空间分布。合格值根据生物膜的深度表示。符号的色彩给人的合规范围。 <img alt="图10" fo:content-width="5in" fo:src="/files/ftp_upload/50857/50857fig10highres.jpg" src="/files/ftp_upload/50857/50857fig10.jpg" /> 图10。磁极蓝图。尺寸给出厘米。 <img alt="图11" fo:content-width="5in" fo:src="/files/ftp_upload/50857/50857fig11highres.jpg" src="/files/ftp_upload/50857/50857fig11.jpg" /> 图11。磁镊子布线。 </str翁&gt;铜管的支持-在A组的尺寸侧视图-用来紧紧缠绕2,120圈铜线，并在它的中心插入软磁合金杆之前建立的电磁线圈（B组 ）。直流电流在线圈中供给所示图C限定磁极性。参见实验概述草图如图4所示。 视频1:该电影只显示注射的细胞-颗粒混合物在毛细管的后生物膜的初始阶段。采集频率为1张/秒，播放短片以10帧/秒。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50857/JoVE_Video1.avi" target="_blank">请点击此处观看该视频。</a> 视频2:这部电影显示经过4小时生长生物膜。颗粒逐渐从分派到B的表面分离<html> iofilm体积，蓝色箭头标记支队事件。采集频率为2图像/分钟和电影播放15帧/秒。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50857/JoVE_Video2.avi" target="_blank">请点击此处观看该视频。</a> 视频3:生物膜后20小时的增长。这部电影是采取位于20微米以上的毛细管底部的中间平面。被收购的2幅图像/分钟频率的图像和电影播放15帧/秒。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50857/JoVE_Video3.avi" target="_blank">请点击此处观看该视频。</a> 视频4:使用ImageJ典型粒子跟踪。这部影片展示了整个序列的起始时间23秒，在时间30秒结束的摘录。黄色的轨迹显示在磁驱动质点位移。采集频率为30帧/秒，播放短片以30帧/秒。https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50857/JoVE_Video4.avi"目标="_blank"&gt;请点击此处观看该视频。

Watch this Scientific Journal Video about Remote Magnetic Actuation of Micrometric Probes for in situ 3D Mapping of Bacterial Biofilm Physical Properties at JoVE.com

Remote Magnetic Actuation of Micrometric Probes for in situ 3D Mapping of Bacterial Biofilm Physical Properties

本文显示了基于接种在细菌生物膜和专用磁镊子的发展原位测量由微生物在接口内置的复杂生命的物质的局部机械特性的磁性粒子的远程致动的原始方法。

测微探针进行远程磁驱动<em&gt;原位</em&gt; 3D细菌生物膜物理性质的映射

Bacterial adhesion and growth on interfaces lead to the formation of three-dimensional heterogeneous structures so-called biofilms. The cells dwelling in ...

remote-magnetic-actuation-micrometric-probes-for-situ-3d-mapping

Nanyang Technological University

Research

JoVE Journal

Bioengineering

Remote Magnetic Actuation of Micrometric Probes for in situ 3D Mapping of Bacterial Biofilm Physical Properties

9.1K 次观看.  Sorbonne Universités, UPMC. 本文显示了基于接种在细菌生物膜和专用磁镊子的发展原位测量由微生物在接口内置的复杂生命的物质的局部机械特性的磁性粒子的远程致动的原始方法。 

视频: Remote Magnetic Actuation of Micrometric Probes for in situ 3D Mapping of Bacterial Biofilm Physical Properties

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches1. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible1. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization1. 
The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart2. It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling3. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc.
Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia14-16.

This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.

多参数的Langendorff灌注兔心脏的光学测绘

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

科研

生物工程

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

在小鼠心脏的动作电位和钙瞬变的光学测绘

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

Magnetically-Assisted Remote Controlled Microcatheter Tip Deflection under Magnetic Resonance Imaging

X-ray fluoroscopy-guided endovascular procedures have several significant limitations, including difficult catheter navigation and use of ionizing radiation, which can potentially be overcome using a magnetically steerable catheter under MR guidance.
	The main goal of this work is to develop a microcatheter whose tip can be remotely controlled using the magnetic field of the MR scanner. This protocol aims to describe the procedures for applying current to the microcoil-tipped microcatheter to produce consistent and controllable deflections.
	A microcoil was fabricated using laser lathe lithography onto a polyimide-tipped endovascular catheter. In vitro testing was performed in a waterbath and vessel phantom under the guidance of a 1.5-T MR system using steady-state free precession (SSFP) sequencing. Various amounts of current were applied to the coils of the microcatheter to produce measureable tip deflections and navigate in vascular phantoms.
	The development of this device provides a platform for future testing and opportunity to revolutionize the endovascular interventional MRI environment.

Current applied to an endovascular microcatheter with microcoil tip made by laser lathe lithography can achieve controllable deflections under magnetic resonance (MR) guidance, which may improve speed and efficacy of navigation of vasculature during various endovascular procedures.

磁力辅助的远程控制微导管尖端偏转根据磁共振成像

X-ray fluoroscopy-guided  endovascular procedures have several significant limitations, including  difficult catheter navigation and use of ionizing ...

Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide nanoparticles (IONP) (Fe3O4) to yield a magnetic bacterial nanocellulose (MBNC). The synthesis of MBNC is a precise and specifically designed multi-step process. Briefly, bacterial nanocellulose (BNC) pellicles are formed from preserved G. xylinus strain according to our experimental requirements of size and morphology. A solution of iron(III) chloride hexahydrate (FeCl3&#183;6H2O) and iron(II) chloride tetrahydrate (FeCl2&#183;4H2O) with a 2:1 molar ratio is prepared and diluted in deoxygenated high purity water. A BNC pellicle is then introduced in the vessel with the reactants. This mixture is stirred and heated at 80 &#176;C in a silicon oil bath and ammonium hydroxide (14%) is then added by dropping to precipitate the ferrous ions into the BNC mesh. This last step allows forming in situ magnetite nanoparticles (Fe3O4) inside the bacterial nanocellulose mesh to confer magnetic properties to BNC pellicle. A toxicological assay was used to evaluate the biocompatibility of the BNC-IONP pellicle. Polyethylene glycol (PEG) was used to cover the IONPs in order to improve their biocompatibility. Scanning electron microscopy (SEM) images showed that the IONP were located preferentially in the fibril interlacing spaces of the BNC matrix, but some of them were also found along the BNC ribbons. Magnetic force microscope measurements performed on the MBNC detected the presence magnetic domains with high and weak intensity magnetic field, confirming the magnetic nature of the MBNC pellicle. Young&#39;s modulus values obtained in this work are also in a reasonable agreement with those reported for several blood vessels in previous studies.

Here, we present a protocol to make a bacterial nanocellulose (BNC) magnetic for applications in damaged blood vessel reconstruction. The BNC was synthesized by G. xylinus strain. On the other hand, magnetization of the BNC was realized through in situ precipitation of Fe2+ and Fe3+ ferrous ions inside the BNC mesh.

一机能磁性纳米纤维素的细菌与氧化铁纳米粒子的制备

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide ...

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances (EPS). The EPS serves as a physical and protective scaffold that houses the bacterial cells and consists of a variety of materials that includes proteins, exopolysaccharides and DNA. The composition of the EPS may change, which remodels the mechanic properties of the biofilm to further develop or support alternative biofilm structures, such as streamers, as a response to environmental cues. Despite this, there are little quantitative descriptions on how EPS components contribute to the mechanical properties and function of biofilms. Rheology, the study of the flow of matter, is of particular relevance to biofilms as many biofilms grow in flow conditions and are constantly exposed to shear stress. It also provides measurement and insight on the spreading of the biofilm on a surface. Here, particle-tracking microrheology is used to examine the viscoelasticity and effective crosslinking roles of different matrix components in various parts of the biofilm during development. This approach allows researchers to measure mechanic properties of biofilms at the micro-scale, which might provide useful information for controlling and engineering biofilms.

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

在生物膜的力学性能原位制图粒子追踪Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

3D胶原凝胶和微通道3D相互作用的研究准备<em&gt;在体内</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan electron-paramagnetic-resonance (RS-EPR), which can provide quantitative information under in vivo conditions on oxygen concentration, pH, redox status and concentration of signaling molecules (i.e., OH&#8226;, NO&#8226;). The RS-EPR technique has a higher sensitivity, improved spatial resolution (1 mm), and shorter acquisition time in comparison to the standard continuous wave (CW) technique. A variety of phantom configurations have been tested, with spatial resolution varying from 1 to 6 mm, and spectral width of the reporter molecules ranging from 16 &#181;T (160 mG) to 5 mT (50 G). A cross-loop bimodal resonator decouples excitation and detection, reducing the noise, while the rapid scan effect allows more power to be input to the spin system before saturation, increasing the EPR signal. This leads to a substantially higher signal-to-noise ratio than in conventional CW EPR experiments.

Rapid Scan Electron Paramagnetic Resonance Opens New Avenues for Imaging Physiologically Important Parameters In Vivo

A new electron paramagnetic resonance (EPR) method, rapid scan EPR (RS-EPR), is demonstrated for 2D spectral spatial imaging which is superior to the traditional continuous wave (CW) technique and opens new venues for in vivo imaging. Results are demonstrated at 250 MHz, but the technique is applicable at any frequency.

快速扫描电子顺磁共振打开新的途径成像生理重要参数<em&gt;在体内</em

We demonstrate a superior method of 2D spectral-spatial imaging of stable radical reporter molecules at 250 MHz using rapid-scan ...

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach recently explored for the development of autologous replacements involves the differentiation of stem cells into chondrocytes. To initiate this chondrogenesis, a degree of compaction of the stem cells is required; hence, we demonstrated the feasibility of magnetically condensing cells, both within thick scaffolds and scaffold-free, using miniaturized magnetic field sources as cell attractors. This magnetic approach was also used to guide aggregate fusion and to build scaffold-free, organized, three-dimensional (3D) tissues several millimeters in size. In addition to having an enhanced size, the tissue formed by magnetic-driven fusion presented a significant increase in the expression of collagen II, and a similar trend was observed for aggrecan expression. As the native cartilage was subjected to forces that influenced its 3D structure, dynamic maturation was also performed. A bioreactor that provides mechanical stimuli was used to culture the magnetically seeded scaffolds over a 21-day period. Bioreactor maturation largely improved chondrogenesis into the cellularized scaffolds; the extracellular matrix obtained under these conditions was rich in collagen II and aggrecan. This work outlines the innovative potential of magnetic condensation of labeled stem cells and dynamic maturation in a bioreactor for improved chondrogenic differentiation, both scaffold-free and within polysaccharide scaffolds.

Chondrogenesis from stem cells requires fine tuning the culture conditions. Here, we present a magnetic approach for condensing cells, an essential step to initiate chondrogenesis. In addition, we show that dynamic maturation in a bioreactor applies mechanical stimulation to the cellular constructs and enhances cartilaginous extracellular matrix production.

3D磁干细胞聚集和生物反应器为成熟软骨再生

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioink Precellularization 无源混合装置打印软骨及皮肤组织类似物的应用

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps compared to standard metallographic sample preparation procedures. The paper proposes a novel bespoke rig for dynamic domain imaging with in situ BH (magnetic hysteresis) measurements and elaborates the protocols for the sensor preparation and the use of the rig to ensure accurate BH measurement. The protocols for static and ordinary dynamic domain imaging (without in situ BH measurements) are also presented. The reported method takes advantage of the convenience and high sensitivity of the traditional Bitter method and enables in situ BH measurement without interrupting or interfering with the domain wall movement processes. This facilitates establishing a direct and quantitative link between the domain wall movement processes&#8211;microstructural feature interactions in ferritic steels with their BH loops. This method is anticipated to become a useful tool for the fundamental study of microstructure&#8211;magnetic property relationships in steels and to help interpret the electromagnetic sensor signals for non-destructive evaluation of steel microstructures.

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample and sensor preparation procedures and the protocols for using the test rig particularly for dynamic domain imaging with in situ BH measurements in order to achieve optimal domain pattern quality and accurate BH measurements.

用原位滞后测量进行磁畴成像的优化设置和协议

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps ...