Department of Chemistry and Biochemistry, Old Dominion University, Norfolk, VA 23529, USA.
Nanoscale. 2012 Apr 28;4(9):2797-812. doi: 10.1039/c2nr11739h. Epub 2012 Feb 13.
Cellular signaling pathways play crucial roles in cellular functions and design of effective therapies. Unfortunately, study of cellular signaling pathways remains formidably challenging because sophisticated cascades are involved, and a few molecules are sufficient to trigger signaling responses of a single cell. Here we report the development of far-field photostable-optical-nanoscopy (PHOTON) with photostable single-molecule-nanoparticle-optical-biosensors (SMNOBS) for mapping dynamic cascades of apoptotic signaling pathways of single live cells in real-time at single-molecule (SM) and nanometer (nm) resolutions. We have quantitatively imaged single ligand molecules (tumor necrosis factor α, TNFα) and their binding kinetics with their receptors (TNFR1) on single live cells; tracked formation and internalization of their clusters and their initiation of intracellular signaling pathways in real-time; and studied apoptotic signaling dynamics and mechanisms of single live cells with sufficient temporal and spatial resolutions. This study provides new insights into complex real-time dynamic cascades and molecular mechanisms of apoptotic signaling pathways of single live cells. PHOTON provides superior imaging and sensing capabilities and SMNOBS offer unrivaled biocompatibility and photostability, which enable probing of signaling pathways of single live cells in real-time at SM and nm resolutions.
细胞信号通路在细胞功能中起着至关重要的作用，也是设计有效治疗方法的关键。不幸的是，细胞信号通路的研究仍然极具挑战性，因为涉及到复杂的级联反应，而且少量分子就足以引发单个细胞的信号反应。在这里，我们报告了远场光稳定光学纳米镜（PHOTON）与光稳定单分子纳米粒子光学生物传感器（SMNOBS）的发展，用于在单个活细胞中单分子（SM）和纳米（nm）分辨率下实时映射细胞凋亡信号通路的动态级联反应。我们已经定量地对单个配体分子（肿瘤坏死因子α，TNFα）及其与受体（TNFR1）的结合动力学进行了成像；实时跟踪其簇的形成和内化及其在细胞内信号通路中的启动；并以足够的时空分辨率研究了单个活细胞的凋亡信号动力学和机制。这项研究为单个活细胞的细胞凋亡信号通路的复杂实时动态级联和分子机制提供了新的见解。PHOTON 提供了卓越的成像和传感功能，SMNOBS 提供了无与伦比的生物相容性和光稳定性，这使得能够在 SM 和 nm 分辨率下实时探测单个活细胞的信号通路。
