Giebel K, Bechinger C, Herminghaus S, Riedel M, Leiderer P, Weiland U, Bastmeyer M
Faculty for Physics, University of Konstanz, D-78457 Konstanz, Germany.
Biophys J. 1999 Jan;76(1 Pt 1):509-16. doi: 10.1016/s0006-3495(99)77219-x.
We have developed a new method for observing cell/substrate contacts of living cells in culture based on the optical excitation of surface plasmons. Surface plasmons are quanta of an electromagnetic wave that travel along the interface between a metal and a dielectric layer. The evanescent field associated with this excitation decays exponentially perpendicular to the interface, on the order of some hundreds of nanometers. Cells were cultured on an aluminum-coated glass prism and illuminated from below with a laser beam. Because the cells interfere with the evanescent field, the intensity of the reflected light, which is projected onto a camera chip, correlates with the cell/substrate distance. Contacts between the cell membrane and the substrate can thus be visualized at high contrast with a vertical resolution in the nanometer range. The lateral resolution along the propagation direction of surface plasmons is given by their lateral momentum, whereas perpendicular to it, the resolution is determined by the optical diffraction limit. For quantitative analysis of cell/substrate distances, cells were imaged at various angles of incidence to obtain locally resolved resonance curves. By comparing our experimental data with theoretical surface plasmon curves we obtained a cell/substrate distance of 160 +/- 10 nm for most parts of the cells. Peripheral lamellipodia, in contrast, formed contacts with a cell substrate/distance of 25 +/- 10 nm.
我们基于表面等离子体激元的光激发，开发了一种观察培养中的活细胞与底物接触的新方法。表面等离子体激元是沿着金属与介电层之间的界面传播的电磁波量子。与这种激发相关的倏逝场垂直于界面呈指数衰减，衰减长度约为几百纳米。细胞培养在镀铝玻璃棱镜上，并从下方用激光束照射。由于细胞会干扰倏逝场，投射到相机芯片上的反射光强度与细胞/底物距离相关。因此，细胞膜与底物之间的接触可以在纳米范围内以高对比度垂直分辨率可视化。沿着表面等离子体激元传播方向的横向分辨率由其横向动量决定，而垂直于该方向时，分辨率由光学衍射极限决定。为了对细胞/底物距离进行定量分析，在不同入射角对细胞进行成像，以获得局部解析的共振曲线。通过将我们的实验数据与理论表面等离子体激元曲线进行比较，我们得出细胞大部分区域的细胞/底物距离为160±10纳米。相比之下，外周片状伪足与细胞底物的接触距离为25±10纳米。
