The development and validation of micro-CT of large deep frozen specimens.

Repetitive freeze/thaw cycles lead to a progressive loss of structural and molecular integrity in deep frozen specimens. The aim of this study was to evaluate a micro-CT stage, which maintains the cryoconservation of large specimens throughout micro-CT imaging. Deep frozen ovine vertebral segments (-20 °C) were fixed in a micro-CT stage made of expanded polystyrene and cooled with dry ice (0 g, 60 g and 120 g). The temperature inside the stage was measured half-hourly over a time span of three hours with subsequent measurement of surface temperature. The method was validated in a series of 30 deep frozen vertebral specimens and in liver tissue after repetitive micro-CT scanning. Isolation without cooling resulted in defrosting. Cooling with 60 g of dry ice led to a temperature rise inside the stage (max. 5.1 °C) and on the specimen surfaces (max. -3 °C). Cooling with 120 g of dry ice resulted in a significant (p < 0.001) and sufficient lowering of the temperature inside the stage (max. -14 °C) and on the surface of the specimens (max. -13.9 °C). The surface temperature during the subsequent micro-CT validation study did not exceed -16 °C (processing time 1 h 45 min). The resolution was 33 μm isotropic voxel side length, enabling a binarization of bone microstructures. Temperature can reliably be maintained below -10 °C during a micro-CT scan by applying the described technique. The resulting spatial resolution and image quality permits a binarization of bone microstructure.