Single-Cell Center, CAS Key Laboratory of Biofuels and Shandong Key Laboratory of Energy Genetics, Qingdao Institute of BioEnergy and Bioprocess Technology, Chinese Academy of Sciences, 189 Songling Road, Qingdao, Shandong 266101, China.
Laboratory for Algae Research and Biotechnology, College of Technology and Innovation, Arizona State University, 7417 E. Unity Avenue, Mesa, Arizona 85212, USA.
Biotechnol Biofuels. 2014 Apr 9;7:58. doi: 10.1186/1754-6834-7-58. eCollection 2014.
Rapid, real-time and label-free measurement of the cellular contents of biofuel molecules such as triacylglycerol (TAG) in populations at single-cell resolution are important for bioprocess control and understanding of the population heterogeneity. Raman microspectroscopy can directly detect the changes of metabolite profile in a cell and thus can potentially serve these purposes.
Single-cell Raman spectra (SCRS) of the unicellular oleaginous microalgae Nannochloropsis oceanica from the cultures under nitrogen depletion (TAG-producing condition) and nitrogen repletion (non-TAG-producing condition) were sampled at eight time points during the first 96 hours upon the onset of nitrogen depletion. Single N. oceanica cells were captured by a 532-nm laser and the SCRS were acquired by the same laser within one second per cell. Using chemometric methods, the SCRS were able to discriminate cells between nitrogen-replete and nitrogen-depleted conditions at as early as 6 hours with >93.3% accuracy, and among the eight time points under nitrogen depletion with >90.4% accuracy. Quantitative prediction of TAG content in single cells was achieved and validated via SCRS and liquid chromatography-mass spectrometry (LC-MS) analysis at population level. SCRS revealed the dynamics of heterogeneity in TAG production among cells in each isogenic population. A significant negative correlation between TAG content and lipid unsaturation degree in individual microalgae cells was observed.
Our results show that SCRS can serve as a label-free and non-invasive proxy for quantitatively tracking and screening cellular TAG content in real-time at single-cell level. Phenotypic comparison of single cells via SCRS should also help investigating the mechanisms of functional heterogeneity within a cellular population.
快速、实时、无需标记地测量生物燃料分子（如三酰基甘油(TAG)）在单细胞分辨率下的细胞含量，对于生物过程控制和理解群体异质性非常重要。拉曼微光谱技术可以直接检测细胞内代谢物谱的变化，因此具有潜在的应用价值。
在氮饥饿（TAG 产生条件）和氮补充（非 TAG 产生条件）培养物中，对单细胞油质微藻 Nannochloropsis oceanica 的单细胞拉曼光谱（SCRS）进行了采样，在氮饥饿开始后的前 96 小时内，每隔 8 小时采集一次。单细胞 N. oceanica 被 532nm 激光捕获，每个细胞在 1 秒内采集 SCRS。通过化学计量学方法，SCRS 能够在氮饥饿开始后 6 小时内以 >93.3%的准确率区分氮饥饿和氮补充条件下的细胞，并且在氮饥饿的 8 个时间点内以 >90.4%的准确率区分。通过 SCRS 和液相色谱-质谱（LC-MS）分析在群体水平上实现了对单个细胞中 TAG 含量的定量预测和验证。SCRS 揭示了每个同基因群体中细胞间TAG 产生异质性的动力学。在单个微藻细胞中观察到 TAG 含量与脂质不饱和程度之间存在显著的负相关。
我们的研究结果表明，SCRS 可作为一种无需标记、非侵入性的方法，用于实时、定量地跟踪和筛选单细胞中的 TAG 含量。通过 SCRS 对单细胞进行表型比较，也有助于研究细胞群体内功能异质性的机制。
