Small biomolecules at the subcellular level are building blocks for the manifestation of complex biological activities. However, non-intrusive in situ investigation of biological systems has been long daunted by the low spatial resolution and poor sensitivity of conventional light microscopies. Traditional infrared (IR) spectro-microscopy can enable label-free visualization of chemical bonds without extrinsic labeling but is still bound by Abbe’s diffraction limit. This review article introduces a way to bypass the optical diffraction limit and improve the sensitivity for mid-IR methods – using tip-enhanced light nearfield in atomic force microscopy (AFM) operated in tapping and peak force tapping modes. Working principles of well-established scattering-type scanning near-field optical microscopy (s-SNOM) and two relatively new techniques, namely, photo-induced force microscopy (PiFM) and peak force infrared (PFIR) microscopy, will be briefly presented. With ∼10-20 nm spatial resolution and monolayer sensitivity, their recent applications in revealing nanoscale chemical heterogeneities in a wide range of biological systems, including biomolecules, cells, tissues, and biomaterials, will be reviewed and discussed. We also envision several future improvements of AFM-based tapping and peak force tapping mode nano-IR methods that permit them to better serve as a versatile platform for uncovering biological mechanisms at the fundamental level.

亚细胞水平的小生物分子是复杂生物活动表现的基石。但是，非侵入式原位长期以来，生物系统的研究一直被传统光学显微镜的低空间分辨率和低灵敏度所吓倒。传统的红外 (IR) 光谱显微镜可以在没有外在标记的情况下实现化学键的无标记可视化，但仍受阿贝衍射极限的限制。这篇评论文章介绍了一种绕过光学衍射极限并提高中红外方法灵敏度的方法——在轻敲和峰值力轻敲模式下使用原子力显微镜 (AFM) 中的尖端增强光近场。将简要介绍成熟的散射型扫描近场光学显微镜 (s-SNOM) 和两种相对较新的技术，即光致力显微镜 (PiFM) 和峰值力红外 (PFIR) 显微镜的工作原理。凭借 10-20 nm 的空间分辨率和单层灵敏度，它们最近在揭示包括生物分子、细胞、组织和生物材料在内的广泛生物系统中的纳米级化学异质性方面的应用将被回顾和讨论。我们还设想了基于 AFM 的轻敲和峰值力轻敲模式纳米红外方法的一些未来改进，使它们能够更好地作为一个多功能平台，在基础层面揭示生物学机制。