*5.3.2.3 Analysis of zymogen granules*

Zymogen granules form a reserve pool of digestive enzymes that are secreted from acinar cells after stimulation. The granules are well visible on TEM images and detecting a change in a number of granules per cell, cumulative granule area per cell, or granule size could suggest a functional change in their physiology. Manual counting of granules is the simplest method to quantify zymogen content; however, it relies on an assumption of typical granule size, and it is a relatively time-consuming approach. Alternatively, manual annotation of individual granules would provide data also on the granule size, but it is an even more time-consuming method that would

*Application of Transmission Electron Microscopy to Detect Changes in Pancreas Physiology DOI: http://dx.doi.org/10.5772/intechopen.104807*

#### **Figure 7.**

*Quantitative analysis of the surface area of zymogen granules. Representative TEM images from CD (A) and WD (E) mice. Image thresholding and watershed separation produce a binary mask (B and E), zymogen granules in black, inset depicts exemplary partitioning of two touching granules). Individual granules are determined using particle analysis on the segmented images (C and G). Panels D and H depict an overlay of the granules on the grayscale TEM image (vesicles in red). Quantification of the granule cross-section area shown in the panel I for a given cell from the CD and the WD mouse. Data were pooled from the following number of ROIs from the CD/WD image: 35/27. Data were analyzed using the Mann–Whitney U test, p values are indicated on graphs.*

effectively hamper analysis or limit it to a few cells only. To at least partially automate the quantification, we took advantage of the fact that zymogen granules have typical properties: (i) granules are spherical structures, and (ii) they appear electron-dense (i.e. dark) on TEM images (**Figure 7A** and **E**).

The following brief instructions describe a useful procedure to partition the granules on a TEM image:


#### **Figure 8.**

*Quantitative analysis of the vacuoles. Representative TEM images from CD (A) and WD (E) mice. Manual annotation of vacuoles resulted in a binary mask (B and E), vacuoles in black; please note that no vacuoles were present in A. Panels C and D depict an overlay of the vacuole binary mask on the grayscale TEM image (vacuoles in red, cytoplasm in green). Percentages of vacuole cross-section areas are shown on the right for the CD (blue) and the WD (red) mouse.*
