Reflected Inline Detection in Epi Oblique Plane Microscopy.

Epi oblique illumination selective plane microscopy (EOPM) facilitates open-top, straightforward sample mounting, enabling high-resolution and high-speed imaging without perturbing biological specimens. However, current EOPM systems typically require three separate microscopes equipped with specialized objectives. This configuration leads to complex and costly setups, significant long-term drift during imaging, reduced numerical aperture, and limited flexibility in adjusting the illumination tilt angle. We introduce Reflected Inline Detection in Epi-Oblique Plane Microscopy (RIDE-OPM), a novel, simplified, and cost-effective design that removes the necessity for a separate inline tertiary microscope and specialized objectives. Our tilt-angle-independent approach leverages the maximum NA of standard objectives, ensuring optimal illumination across the entire field of view without compromising imaging speed or quality, while significantly reducing long-term system drift. The compact and stable RIDE-OPM platform demonstrates robust, unsupervised long-term imaging capabilities. We validated this performance by successfully imaging various biological specimens, including , Drosophila fly brain, human and mouse epithelial cells, and live , underscoring its suitability for advanced long-term biological imaging applications.