Abstract: Release of oil from nuts due to damaged cellular structures can degrade the quality of products incorporating nuts. The aim of this study was to investigate the effects of different processing conditions on microstructure of almond tissue and to quantify these changes using image processing. Spinning disk confocal fluorescence microscopy was used for imaging changes in microstructure of almonds as a function of different thermal processing of almonds. Multiple staining of Nile Red and Calcofluor White was applied to differentiate cell wall structures and oil bodies within individual almond cells without chemical fixation. An algorithm for image processing, included image preprocessing, segmentation, and determination of morphological features of segmented objects, was developed. Oil-roasting processes (140 °C and 150 °C) were found to have a significant impact on microstructure of almonds when compared to the hot air-roasting and blanching processes. Oil-roasted almond at 150 °C had a greater cellular damage due to cell wall and membrane rupture. These changes in microstructure of almonds would make them slightly more susceptible to release oil during storage. The image analysis presented allows quantitative evaluation for the effect of different processing on almond microstructure.