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?...

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?...

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

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 ...

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 ...

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 ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

改良的3D 水凝胶培养的原发胶质细胞的体外Neuroinflammation 模型的建立

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, differentiation, morphology, and molecular synthesis. However, variables such stimuli strength and stimulation times need to be considered when stimulating either cells, tissues or scaffolds. Given that EFs and MFs vary according to cellular response, it remains unclear how to build devices that generate adequate biophysical stimuli to stimulate biological samples. In fact, there is a lack of evidence regarding the calculation and distribution when biophysical stimuli are applied. This protocol is focused on the design and manufacture of devices to generate EFs and MFs and implementation of a computational methodology to predict biophysical stimuli distribution inside and outside of biological samples. The EF device was composed of two parallel stainless-steel electrodes located at the top and bottom of biological cultures. Electrodes were connected to an oscillator to generate voltages (50, 100, 150 and 200 Vp-p) at 60 kHz. The MF device was composed of a coil, which was energized with a transformer to generate a current (1 A) and voltage (6 V) at 60 Hz. A polymethyl methacrylate support was built to locate the biological cultures in the middle of the coil. The computational simulation elucidated the homogeneous distribution of EFs and MFs inside and outside of biological tissues. This computational model is a promising tool that can modify parameters such as voltages, frequencies, tissue morphologies, well plate types, electrodes and coil size to estimate the EFs and MFs to achieve a cellular response.

This protocol describes the step-by-step process to build both electrical and magnetic stimulators used to stimulate biological tissues. The protocol includes a guideline to simulate computationally electric and magnetic fields and manufacture of stimulator devices.

用于刺激生物组织的电动和磁场设备

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, ...