Confinement-sensitive volume regulation dynamics via high-speed nuclear morphological measurements

Diverse tissues in vivo present varying degrees of confinement, constriction, and compression to migrating cells in both homeostasis and disease. The nucleus in particular is subjected to external forces by the physical environment during confined migration. While many systems have been developed to induce nuclear deformation and analyze resultant functional changes, much remains unclear about dynamic volume regulation in confinement due to limitations in time resolution and difficulty imaging in PDMS-based microfluidic chips. Standard volumetric measurement relies on confocal microscopy, which suffers from high phototoxicity, slow speed, limited throughput, and artifacts in fast-moving cells. To address this, we developed a form of double fluorescence exclusion microscopy, designed to function at the interface of microchannel-based PDMS sidewalls, that can track cellular and nuclear volume dynamics during confined migration. By verifying the vertical symmetry of nuclei in confinement, we obtained computational estimates of nuclear surface area. We then tracked nuclear volume and surface area under physiological confinement at a time resolution exceeding 30 frames per minute. We find that during self-induced entrance into confinement, the cell rapidly expands its surface area until a threshold is reached, followed by a rapid decrease in nuclear volume. We next used osmotic shock as a tool to alter nuclear volume in confinement, and found that the nuclear response to hypo-osmotic shock in confinement does not follow classical scaling laws, suggesting that the limited expansion potential of the nuclear envelope might be a constraining factor in nuclear volume regulation in confining environments in vivo.