Mechanisms of surface and volume light scattering from Caenorhabditis elegans revealed by angle-resolved measurements

The roundworm Caenorhabditis elegans has become a widely studied model system in biology in part due to its small size and optical accessibility. Optical imaging of C. elegans primarily relies on scattering of light to generate contrast between the animal and its surroundings, and/or contrast to resolve internal structures within the body. However, the mechanisms of light scattering by C. elegans, and how they influence image contrast, are not well understood, and optimization of contrast is typically performed in a trial-and-error fashion. To address this gap, we performed measurements of light scattering from C. elegans on laboratory substrates under varied illumination angles, wavelength, substrate material, bacterial seeding, and index matching conditions. By combining these results with those of computational simulations, we find that light scattering arises via two components that exhibit distinct angular and spatial distributions: volume scattering within the body and surface scattering at the boundaries between the worm and its surroundings. We elucidated how volume and surface scattering interact with each other and collectively contribute to the overall scattering. Informed by these findings, we determined the illumination angle range, wavelength range, and substrate material that optimize imaging contrast. Our findings provide practical strategies for optimizing image quality across a range of imaging systems.