测微探针进行远程磁驱动<em>原位</em> 3D细菌生物膜物理性质的映射
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Declarações
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Table 1: Reagents and cells | ||||
| Magnetic particles | Life technologies | 14307D | Micrometric magnetic particle, 2.8 µm diameter | |
| Ampicillin (Antibiotic) | Sigma-Aldrich | A9518 | ||
| Tetracycline (Antibiotic) | Sigma-Aldrich | 87128 | ||
| Bacterial strain MG1655gfpF | UGB, Institut Pasteur, France | produces F pili at its surface, resistant to Ampicilllin and tetracycline | ||
| Table 2: Capillaries and tubing | ||||
| Filters for pediatric perfusion | Prodimed-Plastimed | 6932002 | ||
| Hollow Square Capillaries | Composite Metal Scientific | 8280-100 | Manufactured in Borosilicate glass. Square 0.8mm x0.8mm | |
| Tubing silicone peroxyde | VWR international | 228-0512 | Diameter 1mm | |
| Tubing silicone peroxyde | VWR international | 228-0700 | Diameter 3mm | |
| Table 3: Biofilm growth | ||||
| Lysogeny Broth (LB) solution | Amresco-VWR | J106-10PK | standard medium used to grow bacteria | |
| M63B1 solution | Home-made | Standard minimum medium used to grow bacteria | ||
| Glucose | Sigma-Aldrich | G8270 | Used to make M63B1 medium with 0.4% glucose | |
| Table 4: Electronics | ||||
| Camera EMCCD | Hamamatsu | C9100-02 | ||
| Heater controller | World precision instruments | 300354 | ||
| Function generator | Agilent technologies | 33210A | ||
| Power amplifier | Home-made | It gives a current signal with amplitudes up to 4 A. | ||
| Syringe pumps | Kd Scientific | KDS-220 | ||
| Shutter | Vincent Associates | Uniblitz T132 | ||
| Magnetic tweezers | Home-made | 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. | ||
| Table 5: Optics | ||||
| Inverted microscope | Nikon | TE-300 | ||
| S Fluor x40 Objective (NA 0.9, WD0.3) | Nikon | This a long working distance ojective enabling observation of the biofilm in the depth | ||
| Epifluorescence filters: 1) for green fluorescence: Exc 480/20 nm; DM 495; Em 510/20 2) for Red fluorescence: Exc 540/25 nm; DM 565; Em 605/55 | Chroma | 1)#49020 2)#31002 | Particle displacement upon force application is recorded using the red fluoresecnce filter block. | |
| Table 6: Image analysis | ||||
| ImageJ | NIH – particle tracker plugin | |||
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