在生物膜的力学性能原位制图粒子追踪Microrheology

Summary

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.

Abstract

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.

Introduction

大多数细菌细胞能够同时使用浮游（自由生活的），并生长¹面接合（固着）模式。在成长的表面附着模式下，细菌细胞分泌，并包住自己在大量的胞外聚合物（EPS），形成生物膜。所述EPS主要由蛋白质，胞外多糖，胞外DNA，并且对生物膜形成^2。它作为一个物理支架通过该细菌可以用于区分空间上和防止有害的环境条件和宿主反应的细菌。 EPS为不同的组件有生物膜形成³和改变EPS成分的表达不同的角色，可以极大地重塑生物膜结构^4。车部件也可以作为信号分子^5，最近的研究已经显示出一定的EPS部件与微生物细胞相互作用来引导它们的迁移和生物膜的differentiation ^6-8。

研究EPS显着促进了基于由突变体缺陷在EPS ^9,10的特定组件所产生生物膜的形态分析。此外，EPS的特点通常是在宏观尺度（散装特征^）11。形态学分析可以然而缺乏定量细节和散装表征，它返回平均值，失去生物膜的异质性内存在的细节。现在有越来越多的趋势发展到了EPS的力学性能的实时特性，在微观尺度。这个协议演示如何粒子追踪microrheology能够确定的基质组分贝利上的铜绿假单胞菌生物膜⁴的粘弹性和有效交联的时空效果和PSL胞外多糖。

被动microrheology是简单且廉价的rh的eology方法，提供了通过的材料制成的空间微观流变采样的最高迄今为止^12,13。在被动microrheology，探针球体放置在样品中与他们的布朗运动，通过热能量_{（K（B T））}驱动的后跟视频显微镜。几个颗粒可以同时被跟踪，和颗粒的时间依赖性坐标遵循常规随机游走。因此，平均来说，粒子保持在同一位置。然而，位移的标准偏差或均方的颗粒位移（MSD），不为零。由于粘性流体流动，在粘性流体粒子MSD线性增长随着时间的推移。与此相反，聚合物的交联存在于粘弹性或弹性物质有助于他们抵抗流动，颗粒变得有限在他们的位移，导致在MSD曲线（图1A）的高原。这个观察如下的关系MSDαt <s向上>α，其中 ，α是相关的物质的弹性和粘性的贡献率的扩散指数。为粒子朝着粘性流体α= 1，在粘弹性物质0 <α<1，并且在弹性物质α= 0。MSD还可以用于计算蠕变柔量，它是该材料的倾向永久过度变形时间并估计多么容易的材料价差。

颗粒的尺寸，密度和表面化学是微观流变试验的正确应用关键和被选择相对于所研究的系统（在此情况下，生物膜基质的聚合物中，参见图1B）。首先，粒子测量物质与结构，是比颗粒本身小得多的流变性。如果该物质的结构是相似的规模到颗粒，票面的运动视察由各个结构的形状和取向扰动。然而，如果包围粒子的结构是要小得多，这效果小，平均掉，呈现均匀的环境到粒子（图1B）。其次，该粒子的密度应类似于介质（1.05克毫升^-1用于水性介质），使得沉降避免和惯性力是可以忽略不计。大多数颗粒聚苯乙烯晶格符合上述标准。理想的情况下，该颗粒不与生物膜基质的聚合物相互作用作为粒子的MSD的流变解释才有效，如果运动是随机的，具有物质结构从动通过热能和碰撞。这可以通过检查是否在探头颗粒趋于结合或反弹的预生长的生物膜的表面被观察到。然而，尽管缺乏吸引力的生物膜中，颗粒必须能够被并入基质。另外，生物膜的物理化学异质性可能会导致不同的颗粒的更适合作为在生物膜的不同区域的探针。因此，不同大小和表面化学的颗粒应被应用到生物膜。

这样，粒子MSD是能够提供关于组件如何不同向流变性和生物膜的传播有用信息。此外，使用不同的探针的允许一个派生对生物膜的空间异质性理化信息。此方法可用于测试作用抗微生物处理的生物膜的机械性能，或施加到混合物种的生物膜研究如何生物膜的机械性能从引入另一物种的改变。粒子的MSD还可以是用于表征生物膜分散有用。这样的研究将有助于我们生物膜的理解，有可能提高生物膜处理一个生物膜的有益活动的第二工程。

Protocol

1.生物膜栽培细菌菌株制备前1天生物膜培养，接种2毫升合适的生长培养基从冷冻的细菌培养制备的浮游细菌培养物。使用的Luria肉汤培养基（10克L -1 NaCl的10克L- -1酵母提取物，和10g L -1蛋白胨）为粘液P.铜绿假单胞菌和Δ 像素和ΔPSL缺陷的突变体。孵育过夜，在37℃和200 rpm振摇条件。稀释过夜培养物，以使用分光光度计的OD 600的0.40…

Representative Results

The local viscoelastic properties of the biofilm in different regions of the biofilm, which included the voids (medium above the biofilm), plains (undifferentiated flat layer of cells) and microcolonies (see labels in Figure 2A) were investigated. The temporal changes in viscoelastic properties of the biofilm during maturation from days 3 to 5 were also determined. The MSD of the particles in the voids was used as a control and comparable to the MSD of particles in pure medium. In contrast, particles tra…

Discussion

Microrheology is a useful tool for local rheological measurements in heterogeneous systems, such as microbial biofilms. It is a non-destructive technique, enabling the real-time monitoring of rheological changes within the same biological sample over multiple time points. In this protocol, particle-tracking microrheology was applied to Pel and Psl exopolysaccharide mutants in order to investigate how they affect the elasticity and effective crosslinking of the biofilm matrix. Psl favors the development of elastic biofilm…

Materials

Fluorspheres	Invitrogen	F-8821	1.0 um red fluorescent (580/605) microspheres with carboxylate modification
Zeiss Axio Imager M1	Carl Zeiss		Epifluorescent Microscope
Masterflex L/S Digital Drive 07523-80	Cole-Parmer	EW-07523-80	Peristaltic pump
Flow Cell Chambers	Technical University of Denmark
Bubble Trap	Technical University of Denmark
Silicone Tubing	Dow Corning		3 mm outer diameter, 1 mm inner diameter
Clear polypropylene plastic connectors	Cole Parmer	06365-83	1/16 in. (1.588 mm)
Binder Clips			To clamp tubing
Coverslips	Thermo Scientific™ Nunc™		50 x 24 mm
Syringe 3 mL	Terumo
27G Needle	Terumo
2L Storage/Media Bottles	VWR®
Trolley			To hold biofilm setup