**1. Introduction**

The number of people with type 2 diabetes mellitus (T2DM) is growing rapidly worldwide and has already exceeded 530 million in 2021 [1]. Because of the severe consequences of T2DM for patients and the enormous burden on the healthcare system, a lot of research is focused on understanding the development of T2DM. The healthy pancreatic beta cells secrete insulin at a basal rate throughout the day and increase secretion in response to stimulation with nutrients, especially glucose, and other neurohormonal secretagogues, such as acetylcholine and GLP-1, after a meal [2–4]. Understanding the pathophysiology of early phases of glucose tolerance disruption associated with morphological and functional beta cell changes present in insulin-resistant people susceptible to the development of T2DM is especially important and has been the focus of many research groups [5–9]. However, to date very limited information is available about the ultrastructural alterations of beta cells during the early stages of T2DM development [10–13].

Thus, the aim of our chapter is to present the importance and illustrate the usefulness of transmission electron microscopy (TEM) in the research field of pancreas physiology through specific and easily reproducible examples. TEM is a technique used to obtain ultrahigh-resolution images of different samples. The prototype of the transmission electron microscope was developed by Ernst Ruska and Max Knoll in 1931. Since then, TEM has been extensively used in biomedical research, helping us deepen our knowledge about the ultrastructure of cells and understanding the cellular processes. TEM was first used to identify specific features of pancreatic endocrine and exocrine cells in the mid-1950s by Paul Lacy [14] and George Palade [15], respectively. TEM exploits the wavelength properties of electrons to provide greater spatial resolution than the resolution achieved using photons in light microscopy (LM). In TEM, a beam of high-voltage electrons is emitted by the electron gun and then passed through the sample. The most challenging part of TEM is the preparation of samples that are thin and robust enough to allow electrons to penetrate the sample on the one hand and survive the damage caused by the electron beam on the other. In this chapter, TEM is implemented as an appropriate research method that allows us to detect very subtle structural changes in pancreas cells of mice fed with a western diet (WD). Here, we focus on sample preparation, microscopy, and quantitative analysis of ultrastructure, together with some representative results, to illustrate the utility and emphasize the importance of TEM in pancreas research. A comprehensive analysis of ultrastructural changes in WD-fed mice with partly compensated diabetes mellitus will be present in detail elsewhere.

In the first part of this chapter, we briefly summarize the basic anatomical features of the pancreas in humans and mice and describe the main physiological characteristics of the endocrine and exocrine pancreas. Next, we describe the development of T2DM, focusing on the role of obesity and key pathophysiological events. In the central part of this chapter, we discuss the ultrastructural morphology and describe the methodology used for ultrastructural morphometry of the exocrine and endocrine pancreas during the early stages of WD-induced T2DM development.
