Polarization-resolved microscopy for label-free stress-induced birefringence mapping in anisotropic organoids

Conventional optical microscopy has limitations for 3D imaging of thick biological samples or organoids. They also lack the ability to resolve internal stress distributions or provide relative or even absolute mechanical metrics in anisotropic tissues. To address these shortcomings, we have developed a polarization-resolved optical microscopy system to quantify internal stress in anisotropic biological samples. A vertical-cavity surface-emitting laser provides a stable and coherent light source. A birefringent crystal introduces a controllable phase shift between orthogonal polarizations, thereby enhancing anisotropy sensitivity. Two reference photodiodes monitor intensity and phase in real time. The modulated, polarized light illuminates the sample (e.g., a saliva-derived organoid), and stress-induced birefringence alters polarization. The transmitted light is collected by an objective and recorded by a high-resolution industrial camera. Phase retardation is quantitatively analyzed using phase-unwrapping image processing to reconstruct the birefringence distribution and extract the principal stress direction and magnitude. The system detects stress-induced birefringence without the need for staining or labeling. Using the microfluidic chip and culture medium as a baseline, the system exhibited an average background phase noise (standard deviation) of 0.087 ± 0.035 rad, corresponding to a detection limit of approximately 0.262 rad. Based on this foundation, measured phase retardation shifts increased to approximately 0.463 ± 0.151 rad in cell clusters and to 1.217–1.512 rad in ductal regions during growth, indicating stress accumulation that eventually dissipates upon acinar differentiation. These findings reveal distinct spatial variations in stress-induced birefringence. Therefore, this system enables visualization of principal-stress orientation and supports extension to a full 3D optical tomogram via polarization scanning. Finally, this platform offers a label-free, sensitive, and extendable tool for analyzing stress in 3D organoids, soft tissues, and other anisotropic biomaterials.