*5.3.1.1 Normal exocrine and endocrine cell ultrastructure*

The majority of tissue consists of pyramid-shaped exocrine cells (**Figure 2A–C**). These cells form clusters or acini around small ducts and are organized in lobes with thin fibrous tissue. The exocrine cells produce inactive digestive enzymes, seen in the cytoplasm packed in secretory vesicles (**Figure 2C** and **D**), and secrete them into the intercalated ducts which they surround (**Figure 2D**). In each acinus, the exocrine cells are located around the intercalated ducts, with their narrow apical parts oriented to the duct (**Figure 2C** and **D**). The exocrine cells have a round or oval nucleus (**Figure 2C**), located basally. The most prominent structure of the exocrine cells is the rough endoplasmic reticulum (**Figure 2C**) which is present in all different parts of the cell. Numerous round electron-dense secretory vesicles are seen in the perinuclear and apical cytoplasm (**Figure 2C** and **D**). The oval mitochondria (**Figure 2C** and **D**) are found in different parts of the cell.

The endocrine cells are distributed throughout the pancreas (**Figure 2B**) as interlobular positioned clusters of cells termed islets of Langerhans. In the islets of Langerhans (**Figure 2B**) alpha, beta and delta cells can be distinguished (**Figure 2E** and **F**). They are characterized by numerous secretory vesicles. Glucagon granules of alpha cells are large dense-core vesicles and some of them have a pale halo. The core of the granule is of a similar diameter to insulin granules, but the whole granule is smaller by at least 50% (200 nm vs. 350 nm). Insulin-containing vesicles in beta cells are the largest with large clear peripheral halos. Somatostatin-containing granules of delta cells in mice are the smallest and lozenge-shaped (**Figure 2E** and **F**) [93–96]. In the cytoplasm of all types of endocrine cells, abundant rough endoplasmic reticulum, Golgi apparatus, and many oval mitochondria are present (**Figure 2E** and **F**) [97].

### *5.3.1.2 Ultrastructural changes in WD mice*

Comparing the ultrastructure of pancreatic cells in control mice (CD; **Figure 2**) and mice fed the western diet (WD; **Figures 3A** and **B**, **4B**, **5E**, **7E**, and **8D**), we observed many important differences. In the exocrine pancreas of WD mice many necrotic cells (**Figures 3A** and **4B**) are present. In some acinar cells, lipid droplets are seen in the

#### **Figure 3.**

*Mice are fed with western diet (WD). (A) and (B) Ultrathin sections of the pancreas. (A) A necrotic acinar cell containing a lipid droplet in the cytoplasm. (B) Autophagosomes and autolysosomes in the cytoplasm of an acinar cell. AL, autolysosome; AP, autophagosome; L, lipid droplet; M, mitochondrion; N, nucleus; SG, secretory granules; RER, rough endoplasmic reticulum. Scale bars: (A) 2 μm; (B) 500 nm.*

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

cytoplasm (**Figure 3A**). In many cells, numerous autophagic structures, i.e., autophagosomes, autolysosomes (**Figure 3B**), and residual bodies are found. The mitochondria (**Figure 4B**) and rough endoplasmic reticulum (**Figure 5E**) seem to be disorganized, therefore these structures were analyzed in detail.

The structure of the islets of Langerhans in mice fed by WD is non-compact, inhomogeneous, containing more extracellular spaces than in mice fed by CD. There are many necrotic cells in different parts of the islets. In the cytoplasm of endocrine cells, some lipid droplets and many autophagic structures, i.e., autophagosomes, autolysosomes, and residual bodies can be found. Structural differences are also seen in the mitochondria and rough endoplasmic reticulum.

#### *5.3.2 Quantitative analysis of selected cellular structures*

Structural characteristics of mitochondria, rough endoplasmic reticulum, zymogen granules, and vacuoles were studied. To accurately analyze various cell compounds, it is necessary to select TEM images taken at the same magnification.

#### *5.3.2.1 Analysis of mitochondria*

Since mitochondria are crucial for normal beta cell stimulus-secretion coupling and their ultrastructure is altered during the development of T2DM, we analyzed them in more detail. First, we outlined all the mitochondria in the visual field and calculated the surface area in nm<sup>2</sup> . To quantitatively assess the condition of mitochondria, we measured the following shape descriptors: circularity, roundness, aspect ratio of the best fit ellipse, and solidity using Fiji software (NIH) [98].

Circularity (C) is a shape parameter that can mathematically indicate the degree of similarity to a perfect circle. A value of 1.0 indicates a perfect circle. When the circularity value approaches 0.0, the shape becomes less and less circular. Circularity is defined by the equation

#### **Figure 4.**

*Shape descriptors. (A) Circularity versus roundness. (B) Sketch of a mitochondrion (gray) with an overlay of the best fit ellipse (yellow), the major axis of the best fit ellipse (blue), and the minor axis of the best fit ellipse (red). The major axis is used in determining roundness (Eq. (2)) and aspect ratio (Eq. (3)). (C), the shape area (gray, left) and the convex hull area (yellow, right) are used to determine the solidity.*

#### **Figure 5.**

*Analysis of the mitochondria from a CD and a WD mouse. Labeled mitochondria in the image of the exocrine pancreas from the (A) CD and (B) WD mouse. Analysis of the (C) surface area, (D) circularity, (E) roundness, (F) aspect ratio and (G) solidity of an image from CD and WD mouse. Data were pooled from the following number of ROIs from the CD/WD image: 14/16. Data were analyzed using the Mann–Whitney U test, p values are indicated on graphs.*

$$\mathbf{C} = 4\pi \frac{Area}{Perimeter^2} \tag{1}$$

Roundness (R) on the other hand, characterized by

$$R = \frac{4\,\text{Area}}{\pi\,\text{major }\,\text{axis}^2} \tag{2}$$

is similar to circularity, but it is insensitive to irregular borders along the perimeter of the mitochondria and takes into account the major axis of the best fit ellipse. For an illustrative explanation of the differences between circularity and roundness, see **Figure 6A**.

From the best fit ellipse fit to each mitochondrion, the major and minor axes were determined, and the aspect ratio (AR) was calculated by the following equation:

$$AR = \frac{major \ x \text{is}}{minor \ ax \text{is}} \tag{3}$$

AR measures the ratio of an object's height to its width (**Figure 6B**). Therefore, the aspect ratio is equal to one for a perfect circle and increases with an increase in deformation.

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

#### **Figure 6.**

*Quantitative analysis of the RER. Representative TEM images from a CD (A) and a WD (E) mouse. The quantification pipeline involves segmentation (B and F), a binary mask depicting RER cisternae in black and cytosol in white), followed by RER determination using particle analysis on the segmented image (C and G). Overlay of the binary masks on the TEM images (D and H), RER cisternae in red, and cytosol in green). Exemplary percentages of the area covered by the RER cisternae are shown in the top right (CD mouse) and bottom right (WD mouse.*

At the end, solidity (S) was measured using the same Fiji software. Solidity describes the extent to which shape is convex or concave. Taking the area within the mitochondrion and dividing it by the area enclosed by a convex hull provides information about the solidity of the shape (**Figure 6C**). Solidity of a perfectly convex structure is 1, but when the structure becomes more concave, the solidity will deviate from 1. The solidity is defined by

$$S = \frac{area}{conv \times area} \tag{4}$$

In the bullet points that follow, we describe each analysis step in detail.


In **Figure 4** one can observe the results of the above analysis on mitochondria of two representative images of the exocrine pancreas from a CD (**Figure 4A**) and a WD (**Figure 4B**) mouse.
