Raman tweezers sorting of single microbial cells
Raman tweezers sorting of single microbial cells
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DOI：
10.1111/j.1758-2229.2008.00002.x
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发表时间：
2009-02-01
影响因子：
3.3
通讯作者：
Whiteley, Andrew S.
中科院分区：
文献类型：
--
作者：
Huang, Wei E.;Ward, Andrew D.;Whiteley, Andrew S.
我们通过使用拉曼显微光谱和光学捕获相结合的方法来识别并操纵细胞，从而选择性地分离微生物细胞。细胞识别的标准是基于单个细胞的拉曼光谱中的光谱峰位移。苯丙氨酸的峰位从1001到1001的特定移位。厘米(-1)至965版本Cm(-1)用于指示利用(13)C底物的细胞内对(13)C的摄取。使用红外线(1064 Nm)激光捕获和操纵细胞，而使用共排514.5 nm激光在较短的时间尺度(30 S)内获取拉曼光谱。选定的细胞被操纵到毛细管的清洁部分，这些管被切割以物理地分离细胞。该技术在70个单一酵母细胞(酿酒酵母)上进行了细胞活性和交叉污染效应的测试。在这些试验之后，从细菌群中分离出58个表现出摄取(13)C的单一细菌细胞(大肠杆菌DH5α和荧光假单胞菌SBW25：：KM-RFP)。其中18个酵母细胞和7个SBW25：：KM-RFP细胞经孵育回收，7个酵母细胞和3个细菌细胞(SBW25：：KM-RFP)基因组扩增正确。我们发现，拉曼镊子方法有可能为研究不可培养的微生物开辟一个新的前沿，这些微生物占自然环境中微生物总数的99%以上。
We have selectively isolated microbial cells by identifying and then manipulating cells using a combination of Raman microspectroscopy and optical trapping. The criterion for cell discrimination is based on spectral peak shifts within the Raman spectrum of individual cells. A specific shift in the phenylalanine peak position from 1001 rel. cm(-1) to 965 rel. cm(-1) is utilized to indicate the uptake of (13)C within the cell that utilized (13)C-substrate. Cells were captured and manipulated using an infrared (1064 nm) laser while Raman spectra were acquired over shorter timescales (30 s) using a co-aligned 514.5 nm laser beam. Selected cells were manoeuvred to a clean part of a capillary tube and the tubes were cleaved to physically separate the cells. The technique was tested for cell viability and cross-contamination effects using 70 single yeast cells (Saccharomyces cerevisia). Following these tests, 58 single bacterial cells (Escherichia coli DH5 alpha, and Pseudomonas fluorescens SBW25::Km-RFP) that exhibited (13)C uptake were sorted from bacterial populations. Among those isolated cells, 11 out of 18 yeast cells and 7 out of 18 single SBW25:: Km-RFP cells were recovered by incubation; 2 out of 7 sorted yeast cells and 3 out of 8 sorted bacterial cells (single SBW25:: Km-RFP) were genome amplified correctly. We show that the Raman tweezers approach has the potential to open a new frontier to study unculturable microorganisms, which account for more than 99% microbes in natural environment.