Anat Histol Embryol. 2022;51:3–22. wileyonlinelibrary.com/journal/ahe | 3 © 2021 Wiley-VCH GmbH Received: 29 September 2021 | Accepted: 31 October 2021 DOI: 10.1111/ahe.12765 REVIEW Using virtual microscopy for the development of sampling strategies in quantitative histology and design-based stereology Yaroslav Kolinko 1,2 | Anna Malečková 3 | Petra Kochová 3 | Martina Grajciarová 1,2 | Tereza Blassová 1,2 | Tomáš Kural 1 | Andriy Trailin 2 | Lenka Červenková 2,4 | Jiřina Havránková 1,2 | Lucie Vištejnová 1,2 | Pavla Tonarová 2 | Vladimíra Moulisová 2 | Miroslav Jiřík 2,3 | Anna Zavaďáková 2 | Filip Tichánek 2,5 | Václav Liška 2,6 | Milena Králíčková 1,2 | Kirsti Witter 7 | Zbyněk Tonar 1,2 1 Department of Histology and Embryology, Faculty of Medicine in Pilsen, Charles University, Pilsen, Czech Republic 2 Faculty of Medicine in Pilsen, Biomedical Center, Charles University, Pilsen, Czech Republic 3 Faculty of Applied Sciences, European Centre of Excellence NTIS, University of West Bohemia, Pilsen, Czech Republic 4 Department of Pathology, Third Faculty of Medicine, Charles University, Prague, Czech Republic 5 Department of Pathological Physiology, Faculty of Medicine in Pilsen, Charles University, Pilsen, Czech Republic 6 Department of Surgery and Biomedical Center, Faculty of Medicine in Pilsen, Charles University, Pilsen, Czech Republic 7 Institute of Morphology, Department of Pathobiology, University of Veterinary Medicine Vienna, Vienna, Austria Correspondence Zbyněk Tonar, Department of Histology and Embryology, Faculty of Medicine in Pilsen, Charles University, Karlovarská 48, 301 66 Pilsen, Czech Republic. Email: Zbynek.Tonar@lfp.cuni.cz Funding information Horizon 2020 Framework Programme, Grant/Award Number: 856620; Ministerstvo Zdravotnictví Ceské Republiky, Grant/Award Number: 15–29241A and AZV NU20J-08-00009; Ministerstvo Školství, Mládeže a Tělovýchovy, Grant/Award Number: LO1503; Univerzita Karlova v Praze, Grant/Award Number: GACR No. 1313420, Progres Q39, SVV 260 536 and UNCE/MED/006; European Regional Development Fund, Grant/Award Number: AMTMI CZ.02.1.01/0.0/0.0/17_ 048/0007280 and FIND CZ.02.1.01/0.0/0 .0/16_019/0000787 Abstract Only a fraction of specimens under study are usually selected for quantification in histology. Multilevel sampling or tissue probes, slides and fields of view (FOVs) in the regions of interest (ROIs) are required. In general, all parts of the organs under study should be given the same probability to be taken into account; that is, the sampling should be unbiased on all levels. The objective of our study was to provide an over- view of the use of virtual microscopy in the context of developing sampling strategies of FOVs for stereological quantification. We elaborated this idea on 18 examples from multiple fields of histology, including quantification of extracellular matrix and mus- cle tissue, quantification of organ and tumour microvessels and tumour-infiltrating lymphocytes, assessing osseointegration of bone implants, healing of intestine anas- tomoses and osteochondral defects, counting brain neurons, counting nuclei in vitro cell cultures and others. We provided practical implications for the most common situations, such as exhaustive sampling of ROIs, sampling ROIs of different sizes, sam- pling the same ROIs for multiple histological methods, sampling more ROIs with vari- able intensities or using various objectives, multistage sampling and virtual sampling. Recommendations were provided for pilot studies on systematic uniform random sampling of FOVs as a part of optimizing the efficiency of histological quantifica- tion to prevent over- or undersampling. We critically discussed the pros and cons of using virtual sections for sampling FOVs from whole scanned sections. Our review