Interface-Sensitive Epi-Coherent Anti-Stokes Raman Scattering Microscopy for Imaging Cell Adhesion Dynamics

Studying cell adhesion dynamics is critical to understand how cells interact with the extracellular matrix and how they migrate. Conventional microscopy techniques for interfacial studies either lack chemical information or require fluorescence labeling. We found that homodyne epi-coherent anti-Stokes Raman scattering (epi-CARS) microscopy, when focused as interfaces, has intrinsic interfacial selectivity. The interference of the back-reflected nonresonant signals amplifies the contrast of cell adhesion areas, generating a distinctive negative contrast at attachment areas, which reflects the distance between the cell membrane and the substrate with tens of nanometers precision. The Epi-CARS configuration also provides high contrasts for cellular lipid droplets when applied in the inverted microscope configuration. Furthermore, by incorporating a pinhole into our confocal epi-CARS system, we can effectively reject out-of-focus reflections that degrade epi-CARS image quality, allowing us to amplify adhesion and lipid droplet contrasts for complex samples, such as cells. We applied this method to study cell-substrate adhesion dynamics of mitotic cells, revealing adhesion site splitting during mitosis and their outward development during postmitotic spreading. Furthermore, we found that after mitosis, the leading edges exhibited homogeneous adhesion regions with thicker water layers, whereas the retracting edges showed heterogeneous adhesion patterns with thinner layers at the contact sites.