Shibata Takayuki, Furukawa Hiromi, Ito Yasuharu, Nagahama Masahiro, Hayashi Terutake, Ishii-Teshima Miho, Nagai Moeto
Department of Mechanical Engineering, Toyohashi University of Technology, Toyohashi, Aichi 441-8580, Japan.
Department of Mechanical Engineering, Kyushu University, Fukuoka 819-0395, Japan.
Micromachines (Basel). 2020 May 13;11(5):495. doi: 10.3390/mi11050495.
Atomic force microscopy (AFM) is an effective platform for in vitro manipulation and analysis of living cells in medical and biological sciences. To introduce additional new features and functionalities into a conventional AFM system, we investigated the photocatalytic nanofabrication and intracellular Raman imaging of living cells by employing functionalized AFM probes. Herein, we investigated the effect of indentation speed on the cell membrane perforation of living HeLa cells based on highly localized photochemical oxidation with a catalytic titanium dioxide (TiO)-functionalized AFM probe. On the basis of force-distance curves obtained during the indentation process, the probability of cell membrane perforation, penetration force, and cell viability was determined quantitatively. Moreover, we explored the possibility of intracellular tip-enhanced Raman spectroscopy (TERS) imaging of molecular dynamics in living cells via an AFM probe functionalized with silver nanoparticles in a homemade Raman system integrated with an inverted microscope. We successfully demonstrated that the intracellular TERS imaging has the potential to visualize distinctly different features in Raman spectra between the nucleus and the cytoplasm of a single living cell and to analyze the dynamic behavior of biomolecules inside a living cell.
原子力显微镜（AFM）是医学和生物科学中用于活细胞体外操作与分析的有效平台。为了给传统AFM系统引入更多新特性和功能，我们通过使用功能化AFM探针研究了活细胞的光催化纳米加工和细胞内拉曼成像。在此，我们基于用催化二氧化钛（TiO）功能化的AFM探针进行的高度局部光化学氧化，研究了压痕速度对活HeLa细胞膜穿孔的影响。根据压痕过程中获得的力-距离曲线，定量确定了细胞膜穿孔的概率、穿透力和细胞活力。此外，我们在集成了倒置显微镜的自制拉曼系统中，探索了通过用银纳米颗粒功能化的AFM探针进行活细胞内分子动力学的针尖增强拉曼光谱（TERS）成像的可能性。我们成功证明，细胞内TERS成像有潜力清晰地可视化单个活细胞核与细胞质之间拉曼光谱中明显不同的特征，并分析活细胞内生物分子的动态行为。
