Chen De, Shelenkova L, Li Y, Kempf C R, Sabelnikov A
East Carolina University, Greenville, North Carolina 27858, USA.
Anal Chem. 2009 May 1;81(9):3227-38. doi: 10.1021/ac8023476.
The potential of laser tweezers Raman spectroscopy (LTRS) to study complex and dynamic cellular processes was investigated on the model of single E. coli cells lysed (1) from "outside" with egg white lysozyme and (2) from "within" by temperature-induced temperate bacteriophage lambdacI857. The two lysis processes differed in the final outcome (incomplete vs complete cell lysis) as revealed by the dynamic laser light scattering and exhibited distinctive dynamic Raman spectra changes. The technique enabled for the first time at the cellular level to observe and quantify real time interaction of lysozyme with E. coli cells, "visualize" a side effect of the process due to the presence of EDTA, and correlate the process of cell wall disruption, as evidenced by the onset and development of asymmetric speckle scattering patterns, with release/escape of intracellular material (ribosomes, nucleic acids, proteins, etc.) quantified by the intensity changes of Raman signatures. Raman spectra changes observed during the lysis from "within" suggest alleged production of heat shock proteins are consistent with the occurring synthesis of phage-related proteins and are in good agreement with the calculated potential contribution of the above proteins to the Raman spectra. It was also established and validated that the contribution of cellular DNA to the Raman spectra of bacterial cells is negligible compared to RNA. The results open new venues for LTRS research and strongly suggest that LTRS has a great potential especially in investigation of real-time processes.
利用激光镊子拉曼光谱（LTRS）研究复杂且动态的细胞过程的潜力，通过以下两种大肠杆菌单细胞裂解模型进行了研究：（1）用蛋清溶菌酶从“外部”裂解；（2）通过温度诱导的温和噬菌体λcI857从“内部”裂解。如动态激光光散射所示，这两种裂解过程在最终结果上有所不同（不完全细胞裂解与完全细胞裂解），并呈现出独特的动态拉曼光谱变化。该技术首次在细胞水平上能够观察和量化溶菌酶与大肠杆菌细胞的实时相互作用，“可视化”由于存在乙二胺四乙酸（EDTA）而导致的该过程的副作用，并将细胞壁破坏过程（由不对称散斑散射图案的出现和发展证明）与通过拉曼特征强度变化量化的细胞内物质（核糖体、核酸、蛋白质等）的释放/逸出相关联。从“内部”裂解过程中观察到的拉曼光谱变化表明，所谓的热休克蛋白的产生与噬菌体相关蛋白的合成一致，并且与上述蛋白质对拉曼光谱的计算潜在贡献相符。还确定并验证了与RNA相比，细胞DNA对细菌细胞拉曼光谱的贡献可忽略不计。这些结果为LTRS研究开辟了新途径，并强烈表明LTRS具有巨大潜力，特别是在实时过程的研究中。
