Contact-free characterization of nuclear mechanics using correlative Brillouin-Raman Micro-Spectroscopy in living cells

Nuclear mechanics is a key parameter in regulating cell physiology, affecting chromatin accessibility and transcriptional regulation. The most established method to characterize the mechanics of biological materials at the sub-micrometer scale is based on atomic force microscopy (AFM). However, its contact-based nature limits the direct access to the nucleus. While some indirect methods have been proposed to measure nuclear mechanics in living cells, the readout is influenced by the overlaying cytoskeleton. For this reason, mechanical measurements on isolated nuclei are a common strategy to overcome this issue. However, the impact of the invasive preparation procedure on the measured properties is still unclear. To address this issue, we studied the mechanical properties of skin fibroblasts probing the nuclear region and of extracted nuclei using AFM and correlative Brillouin-Raman Micro-Spectroscopy (BRMS). The latter technique is a non-invasive method to image living systems in 3D, obtaining correlative information on the mechanical and chemical properties of the sample at specific points of interest. Using this approach, we demonstrated that extracted nuclei are significantly softer than intact ones. Moreover, we demonstrated the ability of BRMS to highlight mechanical features within living cells that were masked by the convolution with the cytosol in conventional AFM measurements. Overall, this study shows the importance of evaluating nuclear mechanics within the native environment where cellular homeostasis is preserved. We, therefore, suggest that BRMS offers a much deeper insight into nuclear mechanics compared to AFM, and it should be adopted as a reference tool to study nuclear mechanobiology. STATEMENT OF SIGNIFICANCE: The cell nucleus, the largest eukaryotic organelle, is crucial for cellular function and genetic material storage. Its mechanical properties, often altered in disease, influence key processes like chromatin accessibility. Although atomic force microscopy (AFM) is a standard method for studying nuclear mechanics, isolating nuclear stiffness in living cells is challenging due to interference from the cytoskeleton and plasma membrane. We demonstrate that correlative Brillouin-Raman Micro-Spectroscopy (BRMS) enables non-contact, high-resolution measurement of nuclear mechanics, capturing sub-micron details. We compare the results from BRMS with that obtained on the same samples with AFM. BRMS enhances our understanding of nuclear stiffness in physiological conditions, offering valuable insights for researchers in the field of mechanobiology, biotechnology, medicine, and bioengineering.