Enhancement of TIRF Imaging of 3D-Cultured Spheroids via Hydrostatic Compression Using a Balloon Actuator.

Three-dimensional (3D) cultured cells can mimic the in vivo tumor microenvironment more accurately than conventional monolayer cultures. Therefore, they are essential in cancer research and drug discovery. However, high-sensitivity fluorescence imaging of 3D spheroids remains challenging owing to their limited contact with the observation surface and the low penetration depth of total internal reflection fluorescence microscopy (TIRFM). In this study, we developed a microfluidic device equipped with a water-driven balloon actuator that enables the hydrostatic compression of 3D-cultured spheroids. This system gently presses spheroids against a glass surface, significantly enhancing the contact area and improving TIRFM and epifluorescence imaging quality, with more evident improvement observed in TIRFM. Our results show that hydrostatic compression markedly enhances optical accessibility in spheroids while preserving cell viability and structural integrity. The method is designed to complement volumetric imaging techniques, including confocal and light-sheet microscopy, by enabling high-contrast visualization of cell-surface molecular dynamics. Although the current system focuses on surface accessibility, future studies will incorporate rotational mechanisms and automated pressure control to facilitate multi-angle, high-throughput imaging. This platform offers a promising strategy for the dynamic observation of cell-surface interactions in living 3D systems.