Mucus plays an integral role for the barrier function of many epithelial tissues. In the human airways, mucus is constantly secreted to capture inhaled microbes and pollutants and cleared away through concerted ciliary motion. Many important respiratory diseases exhibit altered mucus flowability and impaired clearance, contributing to respiratory distress and increased risk of infections. Understanding how mucus rheology changes during disease progression and in response to treatments is thus of great interest for subtyping patients and tailoring treatments, probing disease mechanisms, and tailoring therapies
however, basic research of mucus rheology is greatly hampered by the lack of scalable and user-friendly rheometry assays for the small volumes of mucus typically produced by in vitro respiratory models and in clinical ex vivo settings. To address this challenge, we developed a streamlined, high-throughput protocol leveraging Differential Dynamic Microscopy (DDM) to reliably measure the frequency-dependent microrheology of minuscule (3-10