The deformation of articular cartilage and its cells at the micro-scale during dynamic activities such as gait has high mechanoregulatory importance. Measuring the cellular geometries during such dynamics has been limited by the rate of microscopic image acquisition. The introduction of resonating mirrors for image rasterization (resonant scanning), rather than the conventional servo control (galvano scanning), has significantly improved the scanning rate by more than 100×. However, the high scanning rate comes at the cost of image quality, thereby posing challenges in image processing. Here, resonance-driven 3-D laser microscopy is used to observe the transient, micro-scale deformation of articular cartilage and its cells under osmotic challenge conditions. Custom image segmentation and deformable registration software were implemented for analysis of the resonance-scanned microscopy data. The software exhibited robust and accurate performance on the osmotic swelling measurements, as well as quantitative validation testing. The resonance-scanning protocol and developed analysis software allow for simultaneous strain calculation of both the local tissue and cells, and are thus a valuable tool for real-time probing of the cell-matrix interactions that are highly relevant in the fields of orthopedic biomechanics, cell mechanobiology, and functional tissue engineering.
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