Live-cell imaging enables reporter-free monitoring of the circadian rhythm in individual Synechocystis cells

In vivo monitoring of circadian rhythms depends on reliable and non-invasive detection methods. This is often achieved by expressing reporter genes heterologously under the control of a circadian promoter. The activity or fluorescence of the gene product is then used as a readout. To avoid the need for generation of such reporter strains, we recently established a reporter-free detection method for cyanobacterial batch cultures. To determine whether these rhythms are driven at the level of individual cells or result from population-based effects, such as gating of cell division, we analyzed individual Synechocystis sp. PCC 6803 cells by combining a microfluidic cultivation technique with multipoint time-lapse microscopy imaging at the single-cell resolution. Hundreds of time-lapse image sequences, acquired over a period of up to ten days, were processed using our deep learning cell segmentation workflow. Although the cells had been entrained by a 12-hour light-dark cycle, neither cell size nor cell division displayed circadian rhythms. This indicates the absence of circadian gating of cell division in Synechocystis. Instead, we observed circadian oscillation in the average brightness of the phase contrast of individual Synechocystis cells. To demonstrate how phase-contrast analysis of single cells can be complemented by backscatter analysis of batch cultures, we investigated the wildtype, a deletion mutant known to affect circadian rhythms (∆kaiC3) and complementation strains at both, the single-cell and batch levels. We concluded that phase contrast and backscatter likely measured the same rhythmic changes in the refractive index of the cells. The method presented here will advance circadian research by enabling the analysis of circadian rhythms in individual cells without the need for expression of reporter molecules.

昼夜节律（circadian rhythms）的体内监测依赖于可靠且无创的检测手段。此类检测通常通过在昼夜节律启动子（circadian promoter）的调控下异源表达报告基因（reporter gene）来实现，随后可将基因产物的活性或荧光信号作为检测读数（readout）。为规避构建此类报告基因菌株的繁琐流程，我们近期开发了一种适用于蓝细菌批量培养物的无报告基因检测方法（reporter-free detection method）。
为探明这些节律是源于单个细胞层面，还是由群体效应（population-based effects，如细胞分裂门控（gating of cell division））所导致，我们结合微流控培养技术（microfluidic cultivation technique）与单细胞分辨率（single-cell resolution）多点延时显微镜成像（multipoint time-lapse microscopy imaging），对单个集胞藻（Synechocystis sp. PCC 6803）细胞展开了分析。我们借助自研的深度学习细胞分割工作流（deep learning cell segmentation workflow），对长达十余天的数百条延时图像序列进行了处理。
尽管这些细胞已通过12小时光暗循环完成了昼夜节律同步化（entrained），但细胞尺寸与细胞分裂均未表现出昼夜节律。这表明集胞藻中不存在细胞分裂的昼夜节律门控现象。与之相反，我们观察到单个集胞藻细胞的相差成像（phase contrast）平均亮度呈现出昼夜振荡现象。
为验证单细胞相差成像分析可通过批量培养物的背散射分析（backscatter analysis）进行补充验证，我们分别在单细胞与批量培养层面，对野生型菌株（wildtype）、已知会影响昼夜节律的ΔkaiC3缺失突变体（∆kaiC3）以及互补菌株（complementation strains）展开了研究。我们得出结论：相差成像与背散射分析大概率检测到了细胞折射率的相同节律性变化。
本研究提出的检测方法无需表达报告分子（reporter molecule），即可实现单个细胞的昼夜节律分析，将推动昼夜节律研究的进一步发展。