Single zonular fiber extraction and characterization: A platform for biomechanical, biochemical and structural analyses.

Zonular fibers are essential for suspending the lens within the eye and, in primates, for transmitting forces generated by the ciliary body to drive ocular accommodation. Despite their biomechanical importance, the response of these fibers to sudden strains, and the molecular basis of their exceptional durability remain poorly understood. This is due, in part, to the absence of tools for studying them in isolation. Here, we present a custom-built, cost-effective apparatus designed to isolate, manipulate, and characterize individual zonular fibers under controlled physical and chemical conditions. The apparatus enables quantification of elastic moduli, stress-relaxation behavior, Poisson's ratio, and response to chemical or enzymatic treatments all from a single fiber, while supporting simultaneous imaging and post-experiment analyses by atomic force microscopy (AFM) or other modalities. We describe the design and calibration of the system, provide protocols for sample mounting and mechanical testing, and introduce accessories for high-resolution imaging and temperature control. Using bovine zonular fibers as a model, we demonstrate the system's ability to capture dynamic responses to enzymatic digestion, including time-resolved degradation kinetics following exposure to α-amylase and trypsin. The modular design, precision, and affordability of the apparatus make it a valuable platform for investigating the mechanics of fine elastic fibers in the eye and beyond, with relevance to connective tissue biology across organ systems.