Measuring strain using digital image correlation of second harmonic generation images

The micromechanical environment of bone is crucial to understanding both bone fracture and mechanobiological responses of osteocytes, yet few techniques exist that are capable of measuring strains on the micrometer scale. A method for measuring micrometer level strains has been developed based on digital image correlation (DIC) of second harmonic generation microscopy (SHGM) images. Bovine tibias milled into thin sections were imaged using SHGM under loads of 0 and 15 MPa. Strains were measured using DIC and compared to applied strain values. First and second principal strains decreased in magnitude as the analysis region area increased from 1750 µm2 to 60,920 µm2, converging to 1.23±0.74 and −0.745±0.9816 times the applied strain respectively. A representative sample histogram revealed regions of pure tensile and compressive strain, and that strains were highly heterogeneous ranging from 8410 to −8840 microstrain for an applied 2870 microstrain. Comparison with applied strain measures suggested that analysis sizes of 1750 µm2 and greater were measuring strains on the tissue scale, and higher resolution is required for collagen fibrillar strains. Regions of low SHGM intensity (“dark” regions) were seen which are believed to be lacunar and perilacunar regions of low collagen density. However, no significant differences in strain magnitude were present in dark regions versus regions of high signal intensity. The proposed technique is effective for strains on the size order of bone microarchitecture, and would be useful for studies into the mechanical microenvironment during loading. The technique also has potential for in vivo studies in small animal models.