[论文解读] Quantum monitoring the metabolism of individual yeast mutant strain cells when aged, stressed or treated with antioxidant

Aryan Morita, Citra Nusantara+9 more|arXiv (Cornell University)|Jul 31, 2020

Diamond and Carbon-based Materials Research参考文献 54被引用 11

一句话总结

本研究利用纳米钻石中氮空位中心的量子钻石磁力计，以亚细胞分辨率检测单个活酵母细胞内的自由基。该技术可实现实时监测衰老、应激及抗氧化剂处理过程中的代谢变化，揭示长寿命突变株tor1Δ和pex19Δ的自由基负荷降低，这或许可解释其寿命延长的原因。

ABSTRACT

Free radicals play a key role in the ageing process. The strongly debated free radical theory of ageing even states that damage caused by free radicals is the main cause of aging on a cellular level. However, free radicals are small, reactive and short lived and thus challenging to measure. We utilize a new technique called diamond magnetometry for this purpose. We make use of nitrogen vacancy centers in nanodiamonds. Via a quantum effect these defects convert a magnetic resonance signal into an optical signal. While this method is increasingly popular for its unprecedented sensitivity in physics, we use this technique here for the first time to measure free radicals in living cells. Our signals are equivalent to T1 signals in conventional MRI but from nanoscale voxels from single cells with sub-cellular resolution. With this powerful tool we are able to follow free radical generation after chemically inducing stress. In addition, we can observe free radical reduction in presence of an antioxidant. We were able to clearly differentiate between mutant strains with altered metabolism. Finally, the excellent stability of our diamond particles allowed us to follow the ageing process and differentiate between young and old cells. We could confirm the expected increase of free radical load in old wild type and sod1Δ mutants. We further applied this new technique to investigate tor1Δ and pex19Δ cells. For these mutants an increased lifespan has been reported but the exact mechanism is unclear. We find a decreased free radical load in, which might offer an explanation for the increased lifespan in these cells.

研究动机与目标

• 利用一种新颖且高度灵敏的检测方法，研究自由基在细胞衰老中的作用。
• 解决在活细胞中以亚细胞空间分辨率测量短寿命、高活性自由基的挑战。
• 区分具有不同衰老表型的突变酵母菌株的代谢应答。
• 评估抗氧化剂对个体细胞内自由基动力学随时间变化的影响。
• 探索tor1Δ和pex19Δ酵母突变株寿命延长的代谢基础。

提出的方法

• 利用纳米钻石中的氮空位（NV）中心作为量子传感器，将磁共振信号转换为光学信号。
• 使用共聚焦显微镜系统检测NV中心的光致发光，该信号反映自由基中未成对电子产生的局部磁场。
• 通过测量NV中心自旋态对自由基生成的响应，实现在活细胞中纳米尺度的检测。
• 进行时间序列测量，追踪衰老、化学应激诱导及抗氧化剂暴露过程中的自由基动力学。
• 比较不同酵母突变株（sod1Δ、tor1Δ、pex19Δ）与野生型细胞的信号强度，评估其代谢差异。
• 利用纳米钻石的高稳定性，对单个细胞进行持续数日的纵向监测。

实验结果

研究问题

• RQ1 在衰老、应激及抗氧化剂处理过程中，单个酵母细胞内的自由基生成如何变化？
• RQ2 量子钻石磁力计能否检测到突变酵母菌株中的亚细胞代谢异质性？
• RQ3 自由基负荷与tor1Δ和pex19Δ酵母突变株寿命延长之间存在何种关系？
• RQ4 与野生型及其他长寿命突变株相比，sod1Δ突变株的代谢特征在氧化应激方面有何差异？
• RQ5 该技术能否在活细胞中以高时间和空间分辨率解析自由基水平的动态变化？

主要发现

• 本研究成功利用纳米钻石中的氮空位中心，以亚细胞分辨率检测到单个酵母细胞内的自由基生成。
• 衰老的野生型及sod1Δ突变株细胞表现出显著升高的自由基负荷，证实了自由基理论衰老假说的预测。
• 抗氧化剂处理导致自由基水平可测量地下调，证明该方法对代谢调节具有高灵敏度。
• 与野生型及sod1Δ菌株相比，tor1Δ和pex19Δ突变株表现出更低的自由基负荷，提示其寿命延长可能与特定代谢机制有关。
• 纵向监测显示年轻与衰老细胞在自由基动力学上存在明显差异，验证了该技术在衰老研究中的实用性。
• 纳米钻石探针表现出优异的光稳定性，可在长时间内重复测量而无信号衰减。