Srinivas S P, Bonanno Joseph A, Larivière Els, Jans Danny, Van Driessche Willy
School of Optometry, Indiana University, Bloomington, IN 47405, USA.
Pflugers Arch. 2003 Oct;447(1):97-108. doi: 10.1007/s00424-003-1145-5. Epub 2003 Aug 21.
Light scattering is an empirical technique employed to measure rapid changes in cell volume. This study describes a new configuration for the method of light scattering and its corroboration by measurements of cell height (as a measure of cell volume). Corneal endothelial cells cultured on glass cover-slips were mounted in a perfusion chamber on the stage of an inverted microscope. A beam of light was focused on the cells from above the stage at an angle of 40 degrees to the plane of the stage. The scattered light intensity (SLI), captured by the objective and referred to as forward light scatter (FLS), increased and decreased in response to hyposmotic and hyperosmotic shocks, respectively. The rapid increase and decrease in SLI corresponded to cell swelling and shrinkage, respectively. Subsequently, SLI decreased and increased as expected for a regulatory volume decrease (RVD) and increase (RVI), respectively. These data are in agreement with measurements of cell height, demonstrating that the method of light scatter in FLS mode is useful for monitoring rapid changes in cell volume of cultured cells. Changes in SLI caused by gramicidin were consistent with cell volume changes induced by equilibration of NaCl and KCl concentrations across the cell membrane. Similarly, an additional decrease in SLI was recorded during RVD upon increasing K+ conductance by valinomycin. Decreasing K+ conductance of the cell membrane with Ba2+ changed the time course of SLI consistent with the effect of the K+ channel blocker on RVD. Bumetanide and dihydro-ouabain inhibited increases in SLI during RVI. In conclusion, FLS is a valid method for qualitative analysis of cell volume changes with a high time resolution.
光散射是一种用于测量细胞体积快速变化的经验技术。本研究描述了光散射方法的一种新配置，并通过测量细胞高度（作为细胞体积的一种度量）对其进行了验证。培养在玻璃盖玻片上的角膜内皮细胞被安装在倒置显微镜载物台上的灌注室中。一束光从载物台上方以与载物台平面成40度角的方向聚焦在细胞上。由物镜捕获并称为前向光散射（FLS）的散射光强度（SLI），分别响应低渗和高渗冲击而增加和减少。SLI的快速增加和减少分别对应于细胞肿胀和收缩。随后，SLI分别如调节性容积减小（RVD）和增加（RVI）所预期的那样下降和上升。这些数据与细胞高度的测量结果一致，表明FLS模式下的光散射方法可用于监测培养细胞的细胞体积快速变化。短杆菌肽引起的SLI变化与细胞膜两侧NaCl和KCl浓度平衡诱导的细胞体积变化一致。同样，在缬氨霉素增加K + 电导时，RVD期间记录到SLI进一步下降。用Ba2 + 降低细胞膜的K + 电导改变了SLI的时间进程，这与K + 通道阻滞剂对RVD的作用一致。布美他尼和二氢哇巴因抑制了RVI期间SLI的增加。总之，FLS是一种具有高时间分辨率的细胞体积变化定性分析的有效方法。
