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

The use of the model roundworm Caenorhabditis elegans has enabled pioneering discoveries in biology, in part due to its optical accessibility. Optical imaging of C. elegans primarily relies on light scattering to generate contrast. However, the mechanisms of light scattering by C. elegans and how they influence image contrast are not well understood. We measured scattering and image contrast from C. elegans under varied illumination angles, wavelength, substrate composition, bacterial seeding, and index matching conditions. Combining experiments with ray-tracing simulations, we show that scattering arises from two interacting components: volume scattering within the body and surface scattering at interfaces between the worm and its surroundings. Surface scattering dominates at low angles and enhances animal boundaries, whereas volume scattering becomes more prominent at higher angles and improves visibility of internal structures. We identify practical conditions that optimize image contrast, including low-angle off-axis illumination, longer-wavelength (red) light, and low-scattering substrate media. Our work establishes a framework for understanding light scattering from small animals.