*5.3.2.4 Analysis of vacuoles*

Cytoplasmic vacuolization is an ultrastructural change associated with pathological alterations in pancreatic cells [101]. The vacuoles appear electron-lucent (i.e., bright) and inhomogeneous on TEM images, very similar to other structures, and this does not allow the use of image thresholding as a technique to effectively detect vacuoles. Since the number of vacuoles per cell is limited, manual annotation of the structures is feasible.

1.Manual detection of the vacuoles: Use Fiji/ImageJ in combination with the builtin ROI manager to outline the vacuoles, which results in a binary mask of the visible structures on the image (**Figure 8B** demonstrating an empty mask, since no vacuoles were visible on the image, and 8E shows several vacuoles that were seen within a cell). Overlaying the structures can serve as visual feedback for quality assessment (**Figure 8C** and **F**).

Quantification of the vacuoles: Use data from the above step to measure the relative area covered by the vacuoles by dividing the cumulative vacuole area by the visible cytoplasm surface. Using this approach, we demonstrated that vacuoles are abundant in the acinar cell of WD treated mice (27% vs. 0% in WD vs. CD mice, **Figure 8** right).
