Simple approach for optical sectioning enhancement in epi-illumination line-scan confocal microscopy

We report the implementation of temporally modulated scanning illumination to enhance optical sectioning in epi-illumination line-scan confocal microscopy. By applying a sinusoidal intensity modulation with a spatial period set to twice the line beam width, and synchronizing the pixel exposure time to the modulation period via rolling shutter control, our method improves the contrast of in-focus signals while effectively suppressing out-of-focus background fluorescence. We demonstrate enhanced sectioning performance through volumetric fluorescence imaging of three samples with different thicknesses: 15 µm fluorescent beads, a ∼30 µm-thick corn grain section, and a ∼180 µm optically cleared mouse kidney. This simple yet practical approach extends the sectioning capability of conventional epi-illumination line-scan confocal systems without requiring hardware modifications, offering a promising solution for high-contrast three-dimensional imaging of thick biological specimens such as organoids.