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 biomedicalresearch. These nanoscale features govern molecular organization, structural dynamics, and cellular function, yet conventional non invasive techniques suchas Fourier-transform infrared spectroscopy (FTIR) are fundamentally limited by optical diffraction. Although hybrid approaches, including scattering-typescanning near-field optical microscopy (s-SNOM) and atomic force microscopy infrared spectroscopy (AFM-IR), have advanced spatial resolution toapproximately 10 nm, this remains insufficient to resolve individual macromolecular assemblies. Furthermore, precise control over the depth of analysis withinbiological architectures, where critical molecular information underpinning intercellular communication resides, has yet to be fully achieved. Here, we applyphoto-induced force microscopy (PiFM), an AFM-based technique that directly measures the force effects of light-induced polarization in the near-field zone,this can be of the order of piconewtons and it is localized perpendicular to the surface thereby enabling a spatial resolution as small as 5 nm at incrementaldepths between 2 nm – 200 nm. Crucially, PiFM can operate under ambient and environmentally controlled conditions, preserving physiologically relevantarchitectures in vitro. Our findings reveal that aldehyde-based fixing, such as formalin treatment, induces significant chemical alterations and spectral overlapin oral mucosa lamina propria progenitor cell (OMLP-PC) nuclear envelopes, underscoring the need for rigorous validation of sample preparation protocols innanospectroscopy. Conversely, live-cell PiFM imaging under controlled humidity enables genuine visualisation of native biomolecular states and dynamicprocesses of OMLP-PC membranes, including extracellular vesicle (EV) biogenesis. PiFM mapping of isolated human bone marrow stromal cell EVs furtheruncovers nanoscale compositional heterogeneity at the single-EV level. This work demonstrates the application of PiFM as a transformative nanospectroscopictool for probing the structural and spatial chemical information of biological matter, potentially down to 5 nm resolution. By bridging physical chemistry andbiophysics, PiFM enables direct visualisation of compositional heterogeneity under near-physiological conditions, offering a non invasive and in situ pathwayfor nanoscale characterisation and mechanistic understanding of biological systems.