Laboratoire Nanotechnologies Nanosystèmes (LN2) - CNRS UMI-3463, Université de Sherbrooke, Canada.
Analyst. 2018 Nov 5;143(22):5559-5567. doi: 10.1039/c8an00911b.
Evanescent field based biosensing systems such as surface plasmon resonance (SPR), diffraction gratings, or metal-clad waveguides (MCWGs) are powerful tools for label-free real-time monitoring of signaling activity of living cells exposed to hormones, pharmacological agents, and toxins. In particular, MCWG-based imaging is well suited for studying relatively thick objects such as cells due to its greater depth of penetration into the sensing medium, compared to SPR. Label-free methods, however, provide only indirect measurements in that the measured signal arises from local changes in material properties rather than from specific biomolecular targets. In the case of cells, the situation is especially complex as the measured label-free signal may result from a combination of very diverse sources: morphological changes, intra-cellular reorganization, cascaded molecular events, protein expression etc. Consequently, deconvolving the contributions of specific sources to a particular cell response profile can be challenging. In the following, we present a cell imaging platform that combines two distinct sensing modalities, namely label-free MCWG imaging and label-based surface enhanced fluorescence (SEF), designed to facilitate the identification of the underlying molecular and structural contributions to the label-free MCWG images. We demonstrate the bimodal capabilities of this imaging platform in experiments designed to visualize actin cytoskeleton organization in vascular smooth muscle cells. We then monitored the real-time response of HEK293 cells expressing the Angiotensin 1 receptor (AT1R), when stimulated by the receptor agonist Angiotensin II (AngII). The analysis of the simultaneous label-free signal obtained by MCWG and the intracellular calcium signal resulting form AT1R activation, measured by SEF, allows relating label-free signal features to specific markers of receptor activation. Our results show that the intracellular calcium levels normally observed following AT1R activation are not required for the initial burst of cellular activity observed in the MCWG signal but rather indicates signaling activity involving the intracellular kinase ROCK.
基于消逝场的生物传感系统，如表面等离子体共振（SPR）、衍射光栅或金属包层波导（MCWG），是用于无标记实时监测激素、药理学试剂和毒素暴露下活细胞信号活性的强大工具。特别是，与 SPR 相比，基于 MCWG 的成像更适合研究相对较厚的物体，如细胞，因为它对传感介质的穿透深度更大。然而，无标记方法仅提供间接测量，因为测量信号源自材料特性的局部变化，而不是特定的生物分子靶标。在细胞的情况下，情况尤其复杂，因为测量的无标记信号可能来自非常不同的来源的组合：形态变化、细胞内重组、级联分子事件、蛋白质表达等。因此，将特定来源的贡献解卷积到特定的细胞反应谱可能具有挑战性。在下面，我们提出了一种细胞成像平台，该平台结合了两种不同的传感模式，即无标记 MCWG 成像和基于标记的表面增强荧光（SEF），旨在促进对无标记 MCWG 图像的基础分子和结构贡献的识别。我们展示了这种成像平台的双模功能，用于可视化血管平滑肌细胞中肌动蛋白细胞骨架组织的实验。然后，我们监测了表达血管紧张素 1 受体（AT1R）的 HEK293 细胞在受受体激动剂血管紧张素 II（AngII）刺激时的实时反应。通过 MCWG 获得的无标记信号和通过 SEF 获得的源自 AT1R 激活的细胞内钙信号的实时响应的分析，使我们能够将无标记信号特征与受体激活的特定标志物相关联。我们的结果表明，在 AT1R 激活后通常观察到的细胞内钙水平对于在 MCWG 信号中观察到的细胞初始活动爆发不是必需的，而是指示涉及细胞内激酶 ROCK 的信号活性。
