Measurement of oxygen concentrations in bacterial biofilms using transient state monitoring by single plane illumination microscopy
Measurement of oxygen concentrations in bacterial biofilms using transient state monitoring by single plane illumination microscopy
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DOI：
10.1088/2057-1976/aa6db7
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发表时间：
2017-06-01
影响因子：
1.4
通讯作者：
Wohland, T.
中科院分区：
文献类型：
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作者：
Karampatzakis, A.;Sankaran, J.;Wohland, T.
需氧细菌的呼吸需要氧气，因此氧气在产生细胞维持和生长所需的能量方面发挥着重要作用。生物膜内氧气的分布可能是不均匀的，并且可能导致细菌细胞根据其局部氧气可用性表现出不同的生理状态。在这里，我们采用了瞬态（TRAST）监测，这是一种非侵入性方法，可以通过时间调制照明来测量三重态的相对数量，以确定生物膜菌落中氧浓度的分布。该方法在可以执行光学切片的单平面照明显微镜上实施，使其成为对厚样品成像的合适方法。我们展示了在铜绿假单胞菌生物膜内以微米分辨率测量的三重态弛豫时间图。我们还提供了一种校准方法，可以根据 TRAST 对比度评估氧气浓度。我们的结果表明，菌落内存在缺氧区，耗氧量延伸到高细胞密度区域之外，建立梯度，直到水平恢复到空气饱和度。
Oxygen is required for respiration in aerobic bacteria and hence plays an essential role in the generation of energy necessary for cell maintenance and growth. The distribution of oxygen within a biofilm can be heterogeneous and may result in bacterial cells exhibiting distinct physiological states depending on their local oxygen availability. Here, we have employed transient state (TRAST) monitoring, a non-invasive method that can measure the relative populations of triplet states by time-modulated illumination, to determine the distribution of oxygen concentrations in biofilm colonies. The method was implemented on a single plane illumination microscope that can perform optical slicing, making it a suitable approach for imaging thick samples. We present a map of triplet relaxation times measured inside a Pseudomonas aeruginosa biofilm at micrometre resolution. We also provide a calibration method that can evaluate oxygen concentration based on TRAST contrast. Our results indicate that anoxic zones exist within the colonies, and oxygen consumption extends outside the areas of high cell densities, establishing a gradient until levels return to air saturation.