Summary

• Volume and surface area of chloroplasts and surface area of plasmodesmata pit fields are presented for two C4 species, maize and sugarcane, with respect to cell surface area and cell volume.
• Serial block face scanning electron microscopy (SBF-SEM) and confocal laser scanning microscopy with the Airyscan system (LSM) were used. Chloroplast size estimates were much faster and easier using LSM than with SBF-SEM; however, the results were more variable than SBF-SEM.
• Mesophyll cells were lobed where chloroplasts were located, facilitating cell-to-cell connections while allowing for greater intercellular airspace exposure. Bundle sheath cells were cylindrical with chloroplasts arranged centrifugally. Chloroplasts occupied c. 30–50% of mesophyll cell volume, and 60–70% of bundle sheath cell volume. Roughly 2–3% of each cell surface area was covered by plasmodesmata pit fields for both bundle sheath and mesophyll cells.
• This work will aid future research to develop SBF-SEM methodologies with the aim to better understand the effect of cell structure on C4 photosynthesis.