The aim: to evaluate combined microscopy techniques for determining the morphological and optical properties of methadone hydrochloride (MDN) crystals. Materials and methods: MDN crystal formation was optimized using a closed container method and crystals were characterized using polarized light microscope (PLM), scanning electron microscopy (SEM) and confocal microscopy (CM). SEM and CM were used to determine MDN crystal thickness and study its relationship with crystal retardation colours using the Michel-Levy Birefringence approach. Results: Dimensions (mean±SD) of diamond shaped MDN crystals were confirmed using SEM and CM. Crystals were 46.4±15.2 Vs 32.0±8.3 µm long, 28.03±8.2 Vs 20.85±5.5 µm wide, and 6.62±2.9 Vs 9.6±4.6 µm thick, respectively. There were significant differences between SEM and CM thickness measurements (U=1283, p<0.05), as the SEM exhibited thinner diamond crystals. The combined use of PLM and Michel-Levy chart enabled the observation of a predominantly yellow coloured MDN crystal, mean thickness at (428 nm) mean retardation value. Conclusion: The SEM was superior and successfully determined MDN crystal dimensions for the first time, whilst the CM results were affected by the Rhodamine dye staining process used for visualisation. The qualitative analysis of the crystallinity status of methadone hydrochloride optimally achieved using a combination of PLM and SEM techniques
