Cell culture methods have been developed in efforts to produce biologically relevant systems for developmental and disease modeling, and appropriate analytical tools are essential. Knowledge of ultrastructural characteristics represents the basis to reveal in situ the cellular morphology, cell-cell interactions, organelle distribution, niches in which cells reside, and many more. The traditional method for 3D visualization of ultrastructural components, serial sectioning using transmission electron microscopy (TEM), is very labor-intensive due to contentious TEM slice preparation and subsequent image processing of the whole collection. In this chapter, we present serial block-face scanning electron microscopy, together with complex methodology for spheroid formation, contrasting of cellular compartments, image processing, and 3D visualization. The described technique is effective for detailed morphological analysis of stem cell spheroids, organoids, as well as organotypic cell cultures.
3D visualization Image reconstruction Image segmentation Morphology Organoid SBF-SEM Scanning electron microscopy Serial block-face Spheroid Stem cell Ultrastructure
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This study was supported by: the project FP7 Regpot ICRC-ERA-HumanBridge no. 316345, the project no. LQ1605 from the National Program of Sustainability II (MEYS CR), Czech Science Foundation (GA16-02702S), the project HistoPARK from the Centre for Analysis and Modeling of Tissues and Organs (CZ.1.07/2.3.00/20.0185, European Social Fund in the Czech Republic), funds from the Faculty of Medicine of Masaryk University (MUNI/M/1050/2013), and the project CEITEC 2020 no. LQ1601 by the Ministry of Education, Youth and Sports of the Czech Republic.
Animation of 3D visualization of the stem cell spheroid (AVI 48109 kb)
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