Fluorescence in situ hybridization (FISH) requires the use of microscopy and imaging technologies to visualize nucleic acids. In addition to analyzing FISH signals, microscopy and specific imaging techniques enable the development of FISH-based approaches for qualitative and quantitative analysis of entire chromosomes and/or chromosomal regions. To avoid repeating textbook descriptions of microscopic fundamentals (including previous editions of this chapter from 2009 to 2017) as well as manufacturer advertising about the performance and applicability of commercially available imaging systems, this chapter focuses on microscopy and imaging in light of FISH-based molecular cytogenetic developments and on the importance and potential of microscopic/digital chromosome visualization for medical genetics education. At present, it seems particularly important to remind/inform biomedical researchers of the chromosomal nature of genome organization, variation (pathogenic variation), and behavior.