Concentration-dependent specimen shrinkage in iodine-enhanced microCT

Iodine potassium iodide (I2 KI) solution can be employed as a contrast agent for the visualisation of soft tissue structures in micro-computed tomography studies. This technique provides high resolution images of soft tissue non-destructively but initial studies suggest that the stain can cause substantial specimen shrinkage. The degree of specimen shrinkage, and potential deformation, is an important consideration when using the data for morphological studies. Here we quantify the macroscopic volume changes in mouse skeletal muscle, cardiac muscle and cerebellum as a result of immersion in the common fixatives 10% phosphate-buffered formal saline, 70% ethanol and 3% glutaraldehyde, compared with I2 KI staining solution at concentrations of 2, 6, 10 and 20%. Immersion in the I2 KI solution resulted in dramatic changes of tissue volume, which were far larger than the shrinkage from formalin fixation alone. The degree of macroscopic change was most dependent upon the I2 KI concentration, with severe shrinkage of 70% seen in solutions of 20% I2 KI after 14 days' incubation. When using this technique care needs to be taken to use the lowest concentration that will give adequate contrast to minimise artefacts due to shrinkage.