Département de Génie Électrique et Génie Informatique, Faculté de Génie, Université de Sherbrooke 2500, Boulevard Université, Sherbrooke, Québec J1K 2R1, Canada.
Biosens Bioelectron. 2013 Dec 15;50:125-31. doi: 10.1016/j.bios.2013.06.018. Epub 2013 Jun 20.
Surface plasmon resonance (SPR) has developed into a powerful approach for label-free monitoring of cellular behavior. Most cellular responses, however, involve a complex cascade of molecular events which makes identifying the specific components of cellular behavior dynamics contributing to the aggregate SPR signal problematic. Recently, a number of groups have used surface plasmon-enhanced fluorescence (SPEF) microscopy on living cells. In this work, we show that SPEF microscopy can be used to identify the molecular mechanisms responsible for SPR detection of cellular processes. By specifically labeling the actin cytoskeleton in human epithelial kidney cells (HEK 293) and rat vascular smooth muscle cells (A7r5), we correlate cell reorganization observed in SPEF with SPR signal variations reflecting aggregate cellular changes. HEK 293 cells stimulated with angiotensin-II exhibited transient contraction, appearing as an SPR signal decrease with a subsequent increase above the initial baseline. SPEF micrographs showed a decrease in cellular area followed by actin densification and cell spreading. A7r5 stimulated with Latrunculin A showed actin cytoskeleton depolymerization, generating a steady SPR signal decrease, with SPEF micrographs showing extensive collapse of cell actin structures. We observed that SPR monitoring of cellular response is strongly dependent on minute variations in cellular footprint on the substrate as well as changes in the molecular density in the basal portions of the cells. Therefore, combining SPR with imaging of selective fluorescent markers by SPEF allows a more comprehensive deconvolution of the cellular signal in relation to molecular events within the cells.
表面等离子体共振（SPR）已发展成为一种用于无标记监测细胞行为的强大方法。然而，大多数细胞反应都涉及到一系列复杂的分子事件，这使得确定导致SPR 信号整体变化的特定细胞行为动力学的组成部分成为一个问题。最近，许多研究小组已经在活细胞上使用了表面等离子体增强荧光（SPEF）显微镜。在这项工作中，我们表明 SPEF 显微镜可用于鉴定导致细胞过程SPR 检测的分子机制。通过在人上皮肾细胞（HEK 293）和大鼠血管平滑肌细胞（A7r5）中特异性标记肌动蛋白细胞骨架，我们将 SPEF 中观察到的细胞重排与反映细胞总体变化的 SPR 信号变化相关联。用血管紧张素 II 刺激的 HEK 293 细胞表现出短暂的收缩，表现为 SPR 信号下降，随后初始基线以上增加。SPEF 显微照片显示细胞面积减小，随后肌动蛋白浓缩和细胞铺展。用拉曲库铵 A 刺激的 A7r5 显示肌动蛋白细胞骨架解聚，产生稳定的 SPR 信号下降，SPEF 显微照片显示细胞肌动蛋白结构广泛崩溃。我们观察到，细胞反应的 SPR 监测强烈依赖于细胞在基底上的足迹的微小变化以及细胞底部分子密度的变化。因此，将 SPR 与 SPEF 对选择性荧光标记物的成像相结合，可以更全面地解析与细胞内分子事件相关的细胞信号。
