Beyond Water Content: Unraveling Stiffness in Hydrated Materials by a Correlative Brillouin–Raman Approach

Brillouin microscopy is revolutionizing bioimaging by enabling noninvasive, label-free mapping of the microscale mechanical properties of biological samples. However, the lack of a robust physical model to disentangle the significant influence of refractive index, density, and, most critically, water content on the Brillouin signal limits its applicability and widespread adoption in complex heterogeneous materials. To address this limitation, a novel Brillouin–Raman correlative microscopy approach is proposed and demonstrated on single cells. In particular, the effects of paraformaldehyde fixation on the morphological, mechanical, and chemical properties of HEK293T cells were investigated. Following fixation, an unexpected decrease in stiffness was observed, accompanied by compositional changes detected via Raman spectroscopy. By modeling cells as biphasic systems, consisting of water and dry mass, the hydration could be decoupled from the stiffness measurements. This approach represents a significant advance in biomechanical analysis, enabling the reliable interpretation of Brillouin data and facilitating the three-dimensional micromechanical characterization of biological materials. Furthermore, it has wide-ranging potential applications in biological research, particularly in contexts where hydration plays a fundamental role, paving the way for novel insights into the diagnosis and analysis of biomedical samples.