Diffusion and binding measurements within oral biofilms using fluorescence photobleaching recovery methods
Diffusion and binding measurements within oral biofilms using fluorescence photobleaching recovery methods
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DOI：
10.1098/rstb.1995.0168
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发表时间：
1995-12-29
期刊：
影响因子：
--
通讯作者：
Treloar, R
中科院分区：
文献类型：
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作者：
Birmingham, JJ;Hughes, NP;Treloar, R
许多研究已经假设，存在于生物膜中的细菌不同于嗜热菌。这些差异被认为会影响生物膜的渗透性，并间接影响生物膜细菌对抗菌剂的敏感性。在这项研究中，使用光漂白后的荧光恢复（FRAP）来监测一组尺寸分级的异硫氰酸荧光素（FITC）缀合的葡聚糖在小区域（约100 nm）上的扩散和结合特性。10 μ m）。根据这些测量结果，可以直接计算表观扩散速率。初步研究的浓度依赖性的葡聚糖相互作用与嗜热菌表明，没有发生不可逆的相互作用，但是，反常的速度比自由溶液的扩散速率。这种现象是使用新的分析和数值方法，结合可逆结合相关的荧光变化建模。生物膜中测得的表观扩散速率高度依赖于生物膜制备。蔗糖饥饿的生物膜产生了明显的减慢2 - 5倍，这取决于葡聚糖分子量和生物膜内的位置，表明生物膜内的扩散受阻。补充蔗糖的生物膜产生的表观扩散速率接近于自由溶液中的表观扩散速率，表明扩散受阻较少。离体菌斑显示出与蔗糖补充的生物膜相似的扩散和结合。FRAP方法为确定细菌生物膜内小面积的扩散速率提供了一种快速方便的方法。这项研究强调了考虑可逆结合和相关荧光变化的影响的重要性，因为这些可能对测得的表观扩散速率有显着影响。
Numerous studies have postulated that bacteria which reside in a biofilm differ from planktonic bacteria. These differences are thought to affect biofilm permeability and, indirectly, the susceptibility of biofilm bacteria to antibacterial agents. In this study fluorescence recovery after photobleaching (FRAP) was used to monitor the diffusion and binding characteristics of a set of size fractionated fluorescein isothiocyanate (FITC)-conjugated dextrans over small areas (ca. 10 mu m) in bacterial biofilms. From these measurements it was straightforward to calculate apparent diffusion rates. Initial studies on the concentration dependence of dextran interaction with planktonic bacteria showed that no irreversible interaction was occurring, however, anomalous faster than free solution diffusion rates were obtained. This phenomenon was modelled using novel analytical and numerical methods which incorporate reversible binding with associated fluorescence changes. Apparent diffusion rates measured in biofilms were highly dependent on biofilm preparation. Sucrose starved biofilms produced an apparent slow-down of two- to fivefold depending on dextran molecular mass and location within the biofilm, indicating that diffusion within the biofilm is hindered. Sucrose supplemented biofilms produced apparent diffusion rates close to those in free solution, suggesting less hindered diffusion. Ex vivo plaque showed diffusion and binding similar to the sucrose supplemented biofilms. The FRAP approach provides a fast and convenient method for determining diffusion rates over small areas within bacterial biofilms. This study reinforces the importance of considering the influence of reversible binding and associated fluorescence changes, as these may have a marked effect on the measured apparent diffusion rate.