The creatine/phosphocreatine system, regulated by Creatine kinase, plays a vital role in maintaining energy balance in the brain. Energy metabolism and the function of creatine kinase are known to be affected in Alzheimer’s diseased brain and cells exposed to the amyloid peptide. In Alzheimer’s disease (AD), the brain and cells that contain amyloid peptide deposits show dysfunctional energy metabolism and impaired creatine kinase activity. Creatine kinase is an important regulator of the creatine/phosphocreatine system that keeps the energy balance in these tissues. Therefore, we hypothesize that an enriched creatine diet provides neuroprotection in early, preclinical AD. Previous publications from our lab indicate increased creatine levels in brain tissue of AD mice. Given that hypo-metabolism likely precedes neurodegeneration and plaque formation, use of creatine to target hypo-metabolism could be a promising approach for altering or decelerating the onset of AD. Infrared spectrochemical imaging was used to examine hippocampal, cortical, and cerebellum tissue from 9-month-old mice expressing triple mutations of PSIM146V, APPSwe and tauP301L transgenes, that progressively develop plaques and tangles. These transgenic mice were used to evaluate the effect of creatine diet (3% w/w) on the development of neurodegeneration. A total of 12 mice (n = 6 background & n = 6 transgenic) were used in this study. These animals were each divided into two groups (n = 3) to be fed regular or creatine-enriched diets. Creatine deposits suggestive of altered energetic status were detected by infrared spectrochemical imaging in all the animals. However, the creatine appearance and crystal morphology was affected by the freeze-thaw procedure involved in sample preparation. Long-term freeze storage also affected creatine crystallization as indicated by a series of freeze storage and freeze-thaw experiments. Creatine crystals can get washed away or migrate from tissue surface during the freeze-thaw procedure as creatine is highly soluble in water. Freeze-thaw experiments confirmed that the sample preparation methods, i.e., methods of tissue acquisition, freezing, storage, and transport affect the formation of the crystals.