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

Davies-Jones, Josh A., Pattem, Jacob K. ORCID: https://orcid.org/0000-0003-0276-7996, Azizova, Liana R., Al-Luaibi, Reem, Morgan, David ORCID: https://orcid.org/0000-0002-6571-5731, Stephens, Phil ORCID: https://orcid.org/0000-0002-0840-4996, Hou, Bo ORCID: https://orcid.org/0000-0001-9918-8223, Clayton, Aled ORCID: https://orcid.org/0000-0002-3087-9226, Nishio, Wayne ORCID: https://orcid.org/0000-0003-2405-1876 and Davies, Philip R. ORCID: https://orcid.org/0000-0003-4394-766X 2026. Unravelling nanoscale chemistries in complex biological systems using photoinduced force microscopy (PiFM). Faraday Discussions 265 , pp. 56-75. 10.1039/d5fd00141b

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Abstract

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.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Schools > Chemistry Professional Services > Vice Chancellors Office Schools > Physics and Astronomy Schools > Dentistry Schools > Medicine Research Institutes & Centres > Cardiff Catalysis Institute (CCI)
Additional Information:	License information from Publisher: LICENSE 1: URL: https://creativecommons.org/licenses/by/3.0/, Start Date: 2026-04-21
Publisher:	Royal Society of Chemistry
ISSN:	1359-6640
Date of First Compliant Deposit:	28 April 2026
Date of Acceptance:	20 February 2026
Last Modified:	02 Sep 2026 10:21
URI:	https://orca.cardiff.ac.uk/id/eprint/186676

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