Reflected Inline Detection in Epi Oblique Plane Microscopy

Abstract

Epi oblique illumination selective plane microscopy (OPM) facilitates open-top, straightforward sample mounting, enabling high-resolution and high-speed imaging without perturbing biological specimens. However, current OPM 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 C. elegans, Drosophila fly brain, human and mouse epithelial cells, and live E. coli, underscoring its suitability for advanced long-term biological imaging applications.