Probing intracellular dynamics in living cells with near-field optics
Probing intracellular dynamics in living cells with near-field optics
复制标题
DOI：
10.1016/s0165-0270(99)00032-1
复制
发表时间：
1999-07-01
影响因子：
3
通讯作者：
Tan, WH
中科院分区：
文献类型：
--
作者：
Bui, JD;Zelles, T;Tan, WH
近场光学(NFO)克服了光学显微镜和光学显微镜的衍射极限，实现了单分子的可视化。然而，尽管NFO在自然科学中的应用很多，但在神经科学中的应用仍然很少。在这项工作中，作者开发了NFO探针来成像活细胞内的动态过程。这是第一次将NFO探头插入活细胞内，将光传递到空间控制的区域进行光学测量，并记录细胞对外部刺激的反应。开发了两种不同的光学检测系统(CCD摄像机和雪崩光子检测)来监测两种不同细胞类型的细胞对药物给药的反应。用NFO探针穿透NG108-15神经母细胞瘤细胞和血管平滑肌细胞(VSMC)。用NFO探针检测药物刺激后细胞内钙离子浓度的变化。用Fura-2/AM或FASH-3/AM钙染料负载细胞。血管紧张素II刺激VSMC，导致精确的细胞内钙浓度升高。在NG108-15细胞中，观察到离子霉素和缓激肽对钙离子升高的不同反应曲线。记录了同一类型的不同细胞之间由于离子霉素引起的反应异质性。结果表明，NFO探针使细胞内事件的实时可视化成为可能。通过改进，细胞内NFO探针提供了探测细胞功能的潜力，具有快速的时间和出色的空间分辨率。(C)1999 Elsevier Science B.V.保留所有权利。
Near-field optics (NFO) overcomes the diffraction limit of light microscopes and pe;mits visualization of single molecules. However, despite numerous applications of NFO in the physical sciences, there is still a paucity of applications in the neurosciences. In this work, the authors have developed NFO probes to image intracellular dynamic processes in living cells. This is the first time a NFO probe has been inserted inside a living cell to deliver light to a spatially controlled region for optical measurements and to record cellular responses to external stimuli. Two different optical detection systems (CCD camera and avalanche photon detection) were developed to monitor cellular responses to drug administration in two different cell types. NG108-15 neuroblastoma cells and vascular smooth muscle cells (VSMC) were penetrated with NFO probes. Intracellular Ca2+ increases post drug stimulation were detected by NFO probes. The cells were loaded with either fura-2/AM or flue-3/AM calcium dyes. VSMC were stimulated with angiotensin II, resulting in a precise area of intracellular Ca2+ increase. Different response profiles of Ca2+ increases were observed after ionomycin and bradykinin administration in NG108-15 cells. Responsive heterogeneities due to ionomycin among different cells of the same type were recorded. The results show that NFO probes make possible real-time visualization of intracellular events. With refinement, intracellular NFO probes offer the potential of probing cell function with fast temporal and excellent spatial resolutions. (C) 1999 Elsevier Science B.V. All rights reserved.