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

自然界中的单细胞细菌通常以附着在固体表面的群落形式生活，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，并且因为它们比自由生活的抗生素更具抗性，所以是一个重大的健康问题。生物膜在生物技术工业中也是不期望的，在生物技术工业中它们可导致管道结垢。为了开发消除生物膜或首先防止它们形成的方法，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随时间形成的生物膜进行成像。使用单光子共聚焦显微镜，可以对生物膜物质的表面和内部细胞成像，但这被限制在约40-50微米或40-50个细胞深，即使在仍然仅允许最大深度为4倍的流动室中也是如此。我们相信，我们现在可以通过使用独特设计的流动室来生长更厚的生物膜，至少达到约200个微生物细胞（200微米）的深度，并通过使用最新的多光子共聚焦激光扫描显微镜（MP-CLSM），使我们能够对这种厚生物膜进行整体成像，从而远远超出这些限制。我们还相信，现在将有可能定位激光，以选择性地杀死生物膜内定义的小区域。如果这是成功的，那么我们将能够评估研究这些区域的再增殖以及这种损伤对生物膜结构完整性的影响的能力。为了研究健康或受损生物膜生物量深处的细胞运动和时间过程，我们将探索可光激活和可光开关荧光蛋白的适用性。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，以评估例如生物膜的抗菌处理的部分功效对其结构完整性及其重新填充受损区域的潜力的结果。这些新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。然而，具体应用不在本项目的范围内。凭借O 'Toole博士领导的技术设施成像部门的专业知识以及货车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，我们在约克设计和评估这些方法和技术方面处于有利地位。

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.