Unravelling nanoscale chemistries in complex biological systems using photoinduced force microscopy (PiFM)

Direct interrogation of nanoscale chemical features on and within biological structures remains a major frontier challenge in biophysical and biomedical research. These nanoscale features govern molecular organization, structural dynamics, and cellular function, yet conventional non-invasive techniques such as Fourier-transform infrared spectroscopy (FTIR) are fundamentally limited by optical diffraction. Although hybrid approaches, including scattering-type scanning near-field optical microscopy (s-SNOM) and atomic force microscopy infrared spectroscopy (AFM-IR), have advanced spatial resolution, they remain insufficient to resolve individual macromolecular assemblies. Furthermore, precise control over the depth of analysis within biological architectures, where critical molecular information underpinning intra- and inter-cellular communication resides, has yet to be fully achieved. Here, we employ photo-induced force microscopy (PiFM), an atomic force microscopy (AFM) based technique that directly measures forces arising from light-induced polarization in the near-field region. These forces, typically on the order of piconewtons, are localized perpendicular to the sample surface. This localization enables a theoretical spatial resolution approaching 5 nm, with depth sensitivity spanning approximately 2-200 nm. Crucially, PiFM can operate under ambient and environmentally controlled conditions, preserving physiologically relevant architectures in vitro. Our findings demonstrate that aldehyde-based fixing, including formalin treatment, causes substantial chemical modifications and spectral overlap within the nuclear envelopes of oral mucosa lamina propria progenitor cells (OMLP-PCs). These effects highlight the necessity for rigorous validation of sample-preparation protocols in nano-spectroscopy. In contrast, live-cell PiFM imaging under controlled humidity conditions enables visualisation of native biomolecular states and dynamic cellular processes in OMLP-PCs. Our approach captured whole-cell and membrane-level phenomena, including extracellular vesicle (EV) biogenesis and nuclear stress responses. PiFM mapping of isolated human bone marrow stromal cell (hBMSC) EVs further uncovers nanoscale compositional heterogeneity at the single-EV level. This work demonstrates the application of PiFM as a transformative nano-spectroscopic tool for probing the structural and spatial chemical information of biological matter, potentially down to 5 nm resolution. By bridging physical chemistry and biophysics, PiFM enables direct visualisation of compositional heterogeneity under near-physiological conditions, offering a non-invasive and in situ pathway for nanoscale characterisation and mechanistic understanding of complex biological systems.