Test-retest reproducibility of atomic force microscopy measurements of human trabecular meshwork stiffness

Purpose: The purpose of the present study was to quantify test-retest reproducibility of measurements of stiffness of the human trabecular meshwork (HTM) by atomic force microscopy (AFM). Methods: Eleven 40 μm radial limbal cryostat sections from a fresh human donor rim were mounted on charged slides and rehydrated at room temperature. Stiffness at four TM locations (anterior to posterior along Schlemm’s canal) was measured by AFM. At each location, a 6 x 6 grid was sampled. Indentation points were evenly distributed over a 20 μm x 20 μm area, with a rate of one load/unload cycle per second. Measurements were then repeated for calculation of test-retest variability. Results: The test-retest coefficients of variation for the four measurement locations (anterior to posterior) were 24.39, 25.28, 12.74, and 14.26%, respectively, with a notable drop in the two posterior locations compared to the anterior. The test-retest coefficient for the sections was 19.17%. For the entire eye, the test-retest coefficient of variation for the measurement of the TM stiffness was 17.13%. Young’s moduli consistently decreased from anterior to posterior location. Conclusions: Wide regional variation suggests that single value does little to fully describe the complex array of TM stiffness levels within the eye, and future studies of TM stiffness assessed by AFM should include multiple tissue samples from each eye, with documentation of the anterior-posterior location of each measurement.