Exploiting advances in imaging in microbiology: methodology development for using a multiphoton laser scanning microscope for biofilm analysis.

利用微生物学成像的进步：使用多光子激光扫描显微镜进行生物膜分析的方法开发。

• 批准号: BB/F003692/1
• 负责人: Marjan Van Der Woude
• 金额: $ 4.45万
• 依托单位: University of York
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2007
• 资助国家: 英国
• 起止时间: 2007 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FF003692%2F1
• 关键词: Exploiting; advances; imaging; microbiology; methodology

Single cell bacteria in nature often live as communities that are attached to a solid surface, which are called biofilms. Similarly, biofilms can form on foreign surfaces in the body, like implants and catheters, and because they are more resistant than their free-living counter parts to antibiotics, are of a significant health concern. Biofilms are also undesirable in the biotechnology industry where they can lead to pipe fouling. To develop ways to eradicate biofilms or prevent them from forming in the first place, we have to understand what is involved in this process. One highly useful approach has been to image a biofilm forming over time. Using a single photon confocal microscopy one can image the surface and the interior cells of the biofilm mass, but this is restricted to around 40-50 micrometer, or 40-50 cells deep, even in flow chambers that still only allow a maximum depth four times that much. We believe we can now go far beyond these limits by using a uniquely designed flow chamber to grow biofilms much thicker, at least up to a depth of approximately 200 microbial cells (200 micrometer) and by using one of the latest multiphoton confocal laser scanning microscopes (MP-CLSM) that should enable us to image this thick biofilm in its totality. We also believe it will now be possible to position the laser to selectively kill defined small regions within the biofilm. If this is successful, we will then be able to assess the ability to study the repopulation of these areas and the effects on biofilm structural integrity of this injury. To study cell movement and temporal processes deep in a healthy or damaged biofilm biomass we will explore the applicability of photactivatable and photoswitchable fluorescent proteins. The more classic techniques of FRAP and FLIP will also be trialed on these thick biofilms using the MP-CLSM. Together, this can become a powerful model system to assess for example the outcome of partial efficacy of antibacterial treatments of a biofilm on its the structural integrity and its potential to repopulate damaged regions. These novel approaches can then be applied to different biological questions and different systems in the future. Specific applications are however outside the scope of this project. We are well-positioned to design and assess these methodologies and technologies at York with the combined expertise of the Technology facility Imaging unit led by Dr. O'Toole, and the extensive experience in molecular microbiology and microbial physiology of Dr. van der Woude and her lab.

自然界中的单细胞细菌通常以群落的形式附着在固体表面上，这种固体表面被称为生物膜。同样，生物膜可以在体内的异物表面形成，如植入物和导管，而且由于它们比自由生活的相应部位对抗生素的抵抗力更强，因此引起了重大的健康问题。生物膜在生物技术工业中也不受欢迎，因为它们可能导致管道污染。为了找到根除生物膜或阻止它们形成的方法，我们必须了解这个过程中涉及的内容。一种非常有用的方法是对生物膜随时间的形成进行成像。使用单光子共聚焦显微镜，人们可以对生物膜团的表面和内部细胞进行成像，但这被限制在40-50微米左右，或40-50个细胞深度，即使在流量室中，也只允许最大深度的四倍。我们相信我们现在可以超越这些限制，通过使用一个独特设计的流动室来生长更厚的生物膜，至少达到大约200个微生物细胞的深度（200微米），并使用最新的多光子共聚焦激光扫描显微镜（MP-CLSM）之一，这应该使我们能够对这种厚的生物膜进行整体成像。我们还相信，现在有可能定位激光，选择性地杀死生物膜内确定的小区域。如果这是成功的，我们将能够评估研究这些地区的重新种群的能力以及这种损伤对生物膜结构完整性的影响。为了研究健康或受损生物膜生物质深处的细胞运动和时间过程，我们将探索光激活和光切换荧光蛋白的适用性。更经典的FRAP和FLIP技术也将使用MP-CLSM在这些厚生物膜上进行试验。总之，这可以成为一个强大的模型系统，例如评估生物膜的结构完整性和重建受损区域的潜力的部分抗菌治疗效果的结果。这些新颖的方法可以在未来应用于不同的生物学问题和不同的系统。但是，特定的应用程序超出了本项目的范围。我们有能力在约克设计和评估这些方法和技术，结合由O'Toole博士领导的技术设施成像单元的专业知识，以及van der Woude博士及其实验室在分子微生物学和微生物生理学方面的丰富经验。

项目成果

Exploiting advances in imaging technology to study biofilms by applying multiphoton laser scanning microscopy as an imaging and manipulation tool.

通过应用多光子激光扫描显微镜作为成像和操作工具，利用成像技术的进步来研究生物膜。

• DOI: 10.1111/j.1365-2818.2009.03190.x
• 发表时间: 2009
• 期刊: Journal of microscopy
• 影响因子: 2
• 作者: Lakins MA

An Atmospheric-Pressure Low-Temperature Plasma Jet for Growth Inhibition of Escherichia Coli

• DOI: 10.1109/tps.2011.2153881
• 发表时间: 2011-06
• 期刊: IEEE Transactions on Plasma Science
• 影响因子: 1.5
• 作者: E. Wagenaars;M. W. van der Woude;R. Vann

CdiA promotes receptor-independent intercellular adhesion.

• DOI: 10.1111/mmi.13114
• 发表时间: 2015-10
• 期刊: Molecular microbiology
• 影响因子: 3.6
• 作者: Ruhe ZC;Townsley L;Wallace AB;King A;Van der Woude MW;Low DA;Yildiz FH;Hayes CS
• 通讯作者: Hayes CS