Hastings Gary, Wang Ruili, Krug Peter, Katz David, Hilliard Julia
Department of Physics and Astronomy, GA State University, Atlanta, GA 30303, USA.
Biopolymers. 2008 Nov;89(11):921-30. doi: 10.1002/bip.21036.
Infrared spectroscopy of biological cell monolayers grown on surfaces is a poorly developed field. This is unfortunate because these monolayers have potential as biological sensors. Here we have used infrared microscopy, in both transmission and transflection geometries, to study air-dried Vero cell monolayers. Using both methods allows one to distinguish sampling artefactual features from real sample spectral features. In transflection experiments, amide I/II absorption bands down-shift 9/4 cm(-1), respectively, relative to the corresponding bands in transmission experiments. In all other spectral regions no pronounced frequency differences in spectral bands in transmission and transflection experiments were observed. Transmission and transflection infrared microscopy were used to obtain infrared spectra for unfixed and acetone- or formalin-fixed Vero cell monolayers. Formalin-fixed monolayers display spectra that are very similar to that obtained using unfixed cells. However, acetone fixation leads to considerable spectral modifications. For unfixed and formalin-fixed monolayers, a distinct band is observed at 1740 cm(-1). This band is absent in spectra obtained using acetone-fixed monolayers. The 1740 cm(-1) band is associated with cellular ester lipids. In support of this hypothesis, two bands at 2925 and 2854 cm(-1) are also found to disappear upon acetone fixation. These bands are associated with C-H modes of the cellular lipids. Acetone fixation also leads to modification of protein amide I and II absorption bands. This may be expected as acetone causes coagulation of soluble cellular proteins. Other spectral changes associated with acetone or formalin fixation in the 1400-800 cm(-1) region are discussed.
表面生长的生物细胞单层的红外光谱分析是一个尚欠发展的领域。这很遗憾，因为这些单层细胞层有作为生物传感器的潜力。在此，我们使用了红外显微镜，采用透射和反射两种几何构型，来研究空气干燥的非洲绿猴肾细胞单层。使用这两种方法能够区分采样伪影特征与真实样品的光谱特征。在反射实验中，酰胺I/II吸收带相对于透射实验中的相应谱带分别下移9/4厘米-1。在所有其他光谱区域，未观察到透射和反射实验中光谱带的明显频率差异。透射和反射红外显微镜被用于获取未固定以及经丙酮或福尔马林固定的非洲绿猴肾细胞单层的红外光谱。福尔马林固定的单层细胞层所显示的光谱与使用未固定细胞获得的光谱非常相似。然而，丙酮固定会导致相当大的光谱改变。对于未固定和福尔马林固定的单层细胞，在1740厘米-1处观察到一个明显的谱带。在使用丙酮固定的单层细胞获得的光谱中不存在这个谱带。1740厘米-1谱带与细胞酯脂有关。为支持这一假设，还发现2925和2854厘米-1处的两个谱带在丙酮固定后消失。这些谱带与细胞脂质的C-H模式有关。丙酮固定还会导致蛋白质酰胺I和II吸收带的改变。这在意料之中，因为丙酮会导致可溶性细胞蛋白质凝固。还讨论了在1400 - 800厘米-1区域与丙酮或福尔马林固定相关的其他光谱变化。
