DropSOAC: Stabilizing Microfluidic Drops for Time-Lapse Quantification of Single-Cell Bacterial Physiology
DropSOAC: Stabilizing Microfluidic Drops for Time-Lapse Quantification of Single-Cell Bacterial Physiology
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DOI：
10.3389/fmicb.2019.02112
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发表时间：
2019-09-24
影响因子：
5.2
通讯作者：
Chang, Connie B.
中科院分区：
文献类型：
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作者：
Pratt, Shawna L.;Zath, Geoffrey K.;Chang, Connie B.
微生物种群中细胞的生理异质性使其对诸如抗菌治疗和营养限制等压力具有韧性。这种恢复力部分归因于能够在这种压力下生存并再生群落的细胞亚群。微流控方法现在提供了一种在单细胞水平上研究微生物生理学和细菌异质性的方法，提高了我们分离和检查这些亚群的能力。基于液滴的微流体提供了一种高通量的方法来研究细菌群体中的单个细胞生理学。采用这种方法，将单个细胞从群体中分离出来，并使用直径15微米的微流控装置将其包裹在分散在油中的生长介质中。液滴在聚二甲基硅氧烷(PDMS)微流控装置内以填充单分子层的形式排列。数以千计的单个细胞在相同的微环境中的生长可以使用共聚焦激光扫描显微镜(CLSM)进行成像。这种方法的一个挑战是在延长的时间推移成像期间保持液滴的稳定性。具体地说，由于流体输送到多孔PDMS环境中，在PDMS设备中孵化期间，液滴不会随时间保持其体积。在这里，我们提出了一种制备PDMS器件的策略，即在微流控芯片上的液滴阵列中稳定液滴位置和体积超过20h，通过将设备浸泡在热力学平衡的含水和油的储存层中来保持油包水液滴的稳定性。这确保了在液滴孵化和成像过程中保持多孔PDMS材料内的液滴乳化液的相平衡。我们展示了这种方法的实用性，我们将其命名为DropSOAC(芯片上的液滴稳定)，用于细菌生长的延时研究。我们研究了铜绿假单胞菌及其Ahpf突变衍生物在复苏期间的生长和饥饿后的生长。我们证明，从单个分离的细胞开始，可以确定数百个单个细菌细胞的生长速度和滞后时间的异质性。结果表明，DropSOAC胶囊为单细胞水平的微生物生理学研究提供了一种高通量的方法，并可用于表征较大种群内细胞的生理差异。
The physiological heterogeneity of cells within a microbial population imparts resilience to stresses such as antimicrobial treatments and nutrient limitation. This resilience is partially due to a subpopulation of cells that can survive such stresses and regenerate the community. Microfluidic approaches now provide a means to study microbial physiology and bacterial heterogeneity at the single cell level, improving our ability to isolate and examine these subpopulations. Drop-based microfluidics provides a high-throughput approach to study individual cell physiology within bacterial populations. Using this approach, single cells are isolated from the population and encapsulated in growth medium dispersed in oil using a 15 mu m diameter drop making microfluidic device. The drops are arranged as a packed monolayer inside a polydimethylsiloxane (PDMS) microfluidic device. Growth of thousands of individual cells in identical microenvironments can then be imaged using confocal laser scanning microscopy (CLSM). A challenge for this approach has been the maintenance of drop stability during extended time-lapse imaging. In particular, the drops do not maintain their volume over time during incubation in PDMS devices, due to fluid transport into the porous PDMS surroundings. Here, we present a strategy for PDMS device preparation that stabilizes drop position and volume within a drop array on a microfluidic chip for over 20 h. The stability of water-in-oil drops is maintained by soaking the device in a reservoir containing both water and oil in thermodynamic equilibrium. This ensures that phase equilibrium of the drop emulsion fluids within the porous PDMS material is maintained during drop incubation and imaging. We demonstrate the utility of this approach, which we label DropSOAC (Drop Stabilization On A Chip), for time-lapse studies of bacterial growth. We characterize growth of Pseudomonas aeruginosa and its Ahpf mutant derivative during resuscitation and growth following starvation. We demonstrate that growth rate and lag time heterogeneity of hundreds of individual bacterial cells can be determined starting from single isolated cells. The results show that the DropSOAC capsule provides a high-throughput approach toward studies of microbial physiology at the single cell level, and can be used to characterize physiological differences of cells from within a larger population.