Optimization of Raman spectra acquisition conditions and its application in a comparison study on mesenchymal stem cells and embryonic stem cells

doi:10.3969/j.issn.2095-4344.2017.17.004

Abstract

Abstract:

BACKGROUND: Raman spectrum, compared with conventional detection technologies, is a rapid, non-invasive and label-free optical method. Its application has become an issue of concern in biomedical research. However, further studies are warranted to optimize the acquisition condition of Raman spectra from different stem cells.
OBJECTIVE: To explore the effect of the wavelength of laser and the groove frequency of gratings to obtain the optimized parameter combination for Raman spectrum collection in human stem cells.
METHODS: Using human mesenchymal stem cells as samples, the effects of different laser wavelengths (532, 638, 785 nm) and different grating groove frequency (600, 1 200, 1 800 gr/mm) on Raman spectra were compared respectively. Then the different combinations of the wavelength and groove frequency were used and compared in terms of the spectra resolution and acquisition time, and the best acquisition condition was selected and applied in a comparison study on the Raman spectra from human mesenchymal stem cells and human embryonic stem cells.
RESULTS AND CONCLUSION: The wavelengths of lasers and groove frequencies of gratings showed compound impacts on both the spikes at different wavenumbers and the ratio between spikes; the combination of 785 nm and 1 200 gr/mm was confirmed to be the best spectrum features for human mesenchymal stem cells. The comparison of Raman spectra from human mesenchymal stem cells and human embryonic stem cells implies that the embryonic stem cells contain higher nucleic acids than the mesenchymal stem cells, while the mesenchymal stem cells appear to contain more proteins and lipids.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Mesenchymal Stem Cells, Embryonic Stem Cells, Spectrum Analysis, Raman, Tissue Engineering

CLC Number:

R394.2

Chen Ming, Cheng Xue-lian, Duan Yong-juan, Hu Xiao. Optimization of Raman spectra acquisition conditions and its application in a comparison study on mesenchymal stem cells and embryonic stem cells[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(17): 2644-2651.

Figures/Tables 3

2.1 激光波长对人间充质干细胞和人胚胎干细胞拉曼光谱中拉曼峰的影响为了验证激光波长对干细胞拉曼光谱的影响，首先比较在不同波长的激光(532，638，785 nm)下人间充质干细胞(图1A-C)与人胚胎干细胞(图1D-F)的拉曼光谱。从图1A与图1D中可以看出3种不同条件下的光谱有多处不同，785 nm条件下光谱在600 cm-1到1 000 cm-1之间具有更清晰的峰型，而532 nm条件下光谱在1 500 cm-1到 1 700 cm-1之间有较高隆起。人间充质干细胞拉曼光谱中1 004 cm-1的平均峰强在3种不同波长激光下依次为基准峰1 450 cm-1的0.74倍、1.15倍和1.65倍(图1B)，人胚胎干细胞拉曼光谱中1 004 cm-1的平均峰强在3种不同波长激光下依次为基准峰1 450 cm-1的0.82倍、1.17倍和1.71倍(图1E)；而人间充质干细胞拉曼光谱中1 660 cm-1的平均峰强相比较于基准峰则依次为1.36倍、1.59倍、0.98倍(图1C)，人胚胎干细胞拉曼光谱中1 660 cm-1的平均峰强相比较于基准峰则依次为1.19倍、1.32倍和0.87倍(图1F)。1 004 cm-1/1 450 cm-1在785 nm波长的激光下比值最大，而1 660 cm-1/1 450 cm-1则在638 nm波长的激光下比值最大。 2.2 光栅刻度密度对人间充质干细胞和人胚胎干细胞拉曼光谱的影响为了验证光栅刻度密度对于干细胞拉曼光谱的影响，进一步比较不同光栅刻度密度(600，1 200， 1 800 gr/mm)条件下人间充质干细胞(图2A-C)和人胚胎干细胞(图2D-F)拉曼光谱的差异。从图2A和图2D中可以看出1 800 gr/mm条件下的光谱在600 cm-1到1 000 cm-1之间相比于1 200 gr/mm和600 gr/mm条件下得到的拉曼光谱具有更加清晰的峰。同时人间充质干细胞拉曼光谱中1 004 cm-1的峰强依次是基准峰1 450 cm-1的0.48倍、0.95倍和1.64倍(图2B)，而1 660 cm-1的峰强则是基准峰1 450 cm-1的0.72倍、0.88倍和0.98倍(图2C)；人胚胎干细胞拉曼光谱中1 004 cm-1的峰强依次是基准峰1 450 cm-1的0.54倍、1.06倍和1.71倍(图2E)，1 660 cm-1的峰强则是基准峰1450 cm-1的0.64倍、0.76倍和0.87倍(图2F)。1 004 cm-1/ 1450 cm-1的均值无论是在人间充质干细胞还是人胚胎干细胞样品中其最大值都出现在1 800 gr/mm的光栅刻度密度条件下，而最小值则出现在600 gr/mm的光栅刻度密度条件下。随着光栅刻度线密度的增加，1 004 cm-1/1 450 cm-1和1 660 cm-1/1 450 cm-1的比值都是逐渐增加的，但是1 004 cm-1/1 450 cm-1的比值增加程度相比于1 660 cm-1/1 450 cm-1的增加程度更明显。 2.3 最佳激光波长和光栅密度光谱收集条件组合的确定在上述工作的基础上，进一步根据光谱分辨率与光谱总收集时间从3种激光波长与3种光栅刻度密度条件组合的9种不同条件(图3A-I)中筛选最佳的一个组合。根据已发表文献中人间充质干细胞的拉曼光谱的重要拉曼峰(在图中用阴影部分区分)在图中是否能较好识别以及组成光谱的数据点数的多少来选择光谱分辨率较高的光谱(表1)[16，23]。从图3A-I中可以看出，光谱在阴影部分的峰位上逐渐变得清晰和突出，其中A-E在阴影部分标注出大部分的峰位表现较为平滑甚至难以识别。根据表1可知只有图3F(638 nm 1 800 gr/mm)、图3H(785 nm，1 200 gr/mm)、图3I(785 nm，1 800 gr/mm)是由超过1 000个数据点构成，这3种光谱相较于其他的光谱在阴影部分具有更加清晰的拉曼峰位，这3种光谱的收集时间分别为100×4 s、100×4 s和100×8 s。 2.4 利用优化的组合条件比较间充质干细胞与胚胎干细胞拉曼光谱胚胎干细胞与间充质干细胞同为重要的干细胞，分别是全能干细胞与多能干细胞，在组织工程上均可作为种子细胞，通过拉曼光谱可以寻找这2种干细胞的内在特性与区别，为干细胞的进一步研究与应用奠定基础。根据上述研究获得的最佳拉曼光谱收集条件 785 nm和1 200 gr/mm，分别收集间充质干细胞与胚胎干细胞的拉曼光谱图，并对结果求均值后得到如图4的差谱图。从图中可以看出，间充质干细胞与胚胎干细胞拉曼光谱的差异主要体现在785 cm-1、811 cm-1、1 095 cm-1、1 575 cm-1等拉曼峰。表2列举了间充质干细胞与胚胎干细胞有显著差异的拉曼光谱峰位，并将人间充质干细胞更强的峰和人胚胎干细胞更强的峰做了区分。 2.5 间充质干细胞与胚胎干细胞拉曼光谱的主成分分析由于间充质干细胞与胚胎干细胞之间在多个波数位置有差异，为了找出这2种干细胞的特征性差异，较好的区分这2种干细胞，为未来拉曼光谱应用于组织工程和干细胞检测提供理论基础，实验借助多元统计学分析方法主成分分析来区分间充质干细胞与胚胎干细胞的拉曼光谱。主成分分析通过降维的思想从众多差异峰位抽提出2个主成分PC1(主成分1)和PC2(主成分2)来代替这些差异，每一张光谱都具有一个PC1和PC2的得分，据此可以将不同的光谱区分开。图5A通过主成分分析抽取出2个主成分PC1(98.6%)和PC2(0.5%)来代替间充质干细胞与胚胎干细胞拉曼光谱多个峰位的差异，从而简化间充质干细胞与胚胎干细胞的区分问题。可以看出利用抽取的前2个主成分可以很好地将间充质干细胞与胚胎干细胞的拉曼光谱区分开，同时，通过图5B的载荷图可以看出影响PC2的主要差异就在 785 cm-1、811 cm-1、1 095 cm-1、1 575 cm-1等峰位。"

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