Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy
Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy
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DOI：
10.3791/3977
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发表时间：
2012-05-01
影响因子：
1.2
通讯作者：
Schenke-Layland, Katja
中科院分区：
文献类型：
--
作者：
Votteler, Miriam;Berrio, Daniel A. Carvajal;Schenke-Layland, Katja
生物医学研究领域需要非破坏性、非接触式和无标签技术来监测细胞和组织培养。然而，目前可用的常规方法需要处理步骤并改变样品的完整性。拉曼光谱是一种快速的方法，可以在不需要进一步处理步骤的情况下测量生物样品。这种基于激光的技术检测单色光的非弹性散射。(6)由于每种化学振动都被分配到特定的拉曼波段（波数以cm（-1）为单位），因此每种生物样品由于其固有的生化成分而具有典型的光谱模式。（7-9）在拉曼光谱中，峰值强度与当前分子键的数量相关。(1)利用多元分析（如主成分分析（PCA））可以检测光谱数据集的相似性和差异性。在这里，我们对活细胞和原生组织进行拉曼光谱分析。在测量之前，细胞要么在玻璃底培养皿中播种，要么在正常细胞培养条件下（37℃，5% CO2）悬浮保存。原生组织被解剖和储存在磷酸盐缓冲盐水（PBS）在4摄氏度之前的测量。根据我们的实验设置，我们要么专注于细胞核，要么专注于细胞外基质（ECM）蛋白质，如弹性蛋白和胶原蛋白。在所有研究中，至少测量30个细胞或30个随机兴趣点。数据处理步骤包括背景减法和归一化。
Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.(1-5) However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.(6) As every chemical vibration is assigned to a specific Raman band (wavenumber in cm(-1)), each biological sample features a typical spectral pattern due to their inherent biochemical composition.(7-9) Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.(1) Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).(10)Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 degrees C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 degrees C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.