Spinning disk confocal microscopy with a 25 Megapixel Camera.

Spinning disk confocal microscopy enables fast optical sectioning with low phototoxicity but is often inaccessible due to high hardware costs. We present a low-cost solution using a 25 megapixel machine vision CMOS camera (Sony IMX540, FLIR Blackfly S) and a custom-built spinning disk. The system uses a back-illuminated sensor with high quantum efficiency (69% at 525 nm) and low read noise (2.31 electrons). High-resolution images of Thy1-GFP mouse brain slices and H&E-stained rat testis verified performance across 3D tissue volumes. The custom disk, made with 18 μm pinholes (180 μm pitch) on a chrome photomask and mounted to an optical chopper motor, enables stable, near-telecentric imaging. Micro-Manager software integration allows synchronized control of all hardware, which demonstrates that affordable CMOS sensors can potentially replace sCMOS in spinning disk microscopy, offering an open-source, scalable solution for advanced imaging.