Second Harmonic Generation Microscopy: A Tool for Spatially and Temporally Resolved Studies of Heat Induced Structural Changes in Meat

Myofibers and collagen show non-linear optical properties enabling imaging using second harmonic generation (SHG) microscopy. The technique is evaluated for use as a tool for real-time studies of thermally induced changes in thin samples of unfixed and unstained pork. The forward and the backward scattered SHG light reveal complementary features of the structures of myofibers and collagen fibers. Upon heating the myofibers show no structural changes before reaching a temperature of 53 °C. At this temperature the SHG signal becomes extinct. The extinction of the SHG at 53 °C coincides with a low-temperature endotherm peak observable in the differential scanning calorimetry (DSC) thermograms. DSC analysis of epimysium, the connective tissue layer that enfold skeletal muscles, produces one large endotherm starting at 57 °C and peaking at 59.5 °C. SHG microscopy of collagen fibers reveals a variability of thermal stability. Some fibers show severe shrinkage at 57 °C, before the signal for most of them vanishes between 59 °C and 61 °C and thus coinciding with the endotherm of the thermograms. However, in some areas, strong SHG signals from collagen can be visualized even after prolonged heating to 67 °C and thus indicating regions of much higher thermal stability. It is seen that the benefits of the structural and temporal information available from SHG microscopy reveals complementary information to a traditional DSC measurement and enables a more complete understanding of the thermal denaturation process.