Effect of Laser Irradiation on Cell Function and Its Implications in Raman Spectroscopy.
Effect of Laser Irradiation on Cell Function and Its Implications in Raman Spectroscopy.
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DOI：
10.1128/aem.02508-17
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发表时间：
2018-04-15
影响因子：
4.4
通讯作者：
Yin H
中科院分区：
文献类型：
--
作者：
Yuan X;Song Y;Song Y;Xu J;Wu Y;Glidle A;Cusack M;Ijaz UZ;Cooper JM;Huang WE;Yin H
激光在先进的生物成像和拉曼光谱中发挥着重要作用。然而，它们也因其对生物的破坏性影响而广为人知，这导致了人们对激光技术不利影响的担忧。为了实现拉曼光谱用于细胞分析和操作，例如拉曼激活的细胞分选，识别活细胞的非破坏性条件是至关重要的。在这里，我们在单细胞水平上定量评估了532 nm激光照射对细菌细胞命运和生长的影响。使用专门建造的微流控平台，我们能够量化生长特征，即单个细胞的特定生长率和滞后时间，以及结合拉曼光谱的群体存活率。具有代表性的革兰氏阴性和革兰氏阳性物种对激光照射剂量的反应显示出类似的趋势。激光照射可损害细胞的生理功能，破坏程度与剂量和应变有关，从细胞生长减慢到细胞代谢活性完全丧失，最后到物理解体。革兰氏阳性细菌细胞比革兰氏阴性细菌更容易受到辐射损伤。通过直接将拉曼采集与单细胞生长特性相关联，我们提供了单个细菌细胞的拉曼光谱无损特性的证据。然而，虽然可以在不导致细胞死亡的情况下获得强大的拉曼信号，但如果细胞活力至关重要，来自不同菌株和单个细胞的不同响应证明了仔细评估拉曼获取条件的合理性。在拉曼光谱中的重要性，在基于激光的系统中使用强大的单色光有助于检测固有的微弱信号。这使得可以在单细胞水平上测量与环境和临床相关的微生物。能够进行拉曼测量的意义在于，与基于标记的荧光技术不同，它提供了一个特定于任何(未标记的)样品的身份和状态的“指纹”。因此，它已经成为研究生理和环境条件下活细胞的一种强有力的方法。然而，激光的高功率也有可能杀死细菌，这引发了人们的担忧。这项研究是一项定量评估，为评估激光照射对单个细菌细胞的影响提供了一个通用的平台和方法。此外，它通过确定非破坏性测量来自几个不同组的代表性细菌的光谱所需的条件来说明这一点。
Lasers are instrumental in advanced bioimaging and Raman spectroscopy. However, they are also well known for their destructive effects on living organisms, leading to concerns about the adverse effects of laser technologies. To implement Raman spectroscopy for cell analysis and manipulation, such as Raman-activated cell sorting, it is crucial to identify nondestructive conditions for living cells. Here, we evaluated quantitatively the effect of 532-nm laser irradiation on bacterial cell fate and growth at the single-cell level. Using a purpose-built microfluidic platform, we were able to quantify the growth characteristics, i.e., specific growth rates and lag times of individual cells, as well as the survival rate of a population in conjunction with Raman spectroscopy. Representative Gram-negative and Gram-positive species show similar trends in response to a laser irradiation dose. Laser irradiation could compromise the physiological function of cells, and the degree of destruction is both dose and strain dependent, ranging from reduced cell growth to a complete loss of cell metabolic activity and finally to physical disintegration. Gram-positive bacterial cells are more susceptible than Gram-negative bacterial strains to irradiation-induced damage. By directly correlating Raman acquisition with single-cell growth characteristics, we provide evidence of nondestructive characteristics of Raman spectroscopy on individual bacterial cells. However, while strong Raman signals can be obtained without causing cell death, the variety of responses from different strains and from individual cells justifies careful evaluation of Raman acquisition conditions if cell viability is critical. IMPORTANCE In Raman spectroscopy, the use of powerful monochromatic light in laser-based systems facilitates the detection of inherently weak signals. This allows environmentally and clinically relevant microorganisms to be measured at the single-cell level. The significance of being able to perform Raman measurement is that, unlike label-based fluorescence techniques, it provides a “fingerprint” that is specific to the identity and state of any (unlabeled) sample. Thus, it has emerged as a powerful method for studying living cells under physiological and environmental conditions. However, the laser's high power also has the potential to kill bacteria, which leads to concerns. The research presented here is a quantitative evaluation that provides a generic platform and methodology to evaluate the effects of laser irradiation on individual bacterial cells. Furthermore, it illustrates this by determining the conditions required to nondestructively measure the spectra of representative bacteria from several different groups.