Confocal measurement of the three-dimensional size and shape of plant parenchyma cells in a developing fruit tissue

Parenchyma cells from the inner mesocarp of a grape berry (Vitis vinifera L , cv. Chardonnay) were visualised in three-dimensions within a whole mount of cleared, stained tissue using confocal laser scanning microscopy and dig ital image reconstruction. The whole berry was fixed, bisected longitudinal ly, cleared in methyl salicylate, stained with safranin O and mounted in me thyl salicylate. Optical slices were collected at 1.0 mu m intervals to a d epth of 150 mu m. Neighbouring z-series were joined post-collection to doub le the field-of-view. Attenuation at depth of the fluorescent signal from c ell walls was quantified and corrected. Axial distortion due to refractive index mismatch between the immersion and mounting media was calibrated usin g yellow-green fluorescent microspheres and corrected. Transmission electro n microscopy was used to correct fluorescent measurements of cell wall thic kness. Digital image reconstructions of wall-enclosed spaces enabled cells to be rendered as geometric solids of measurable surface area and volume. C ell volumes within the inner mesocarp tissue of a single grape berry exhibi ted a 14-fold range, with polysigmoidal distribution and groupings around s pecific size classes. Cell shape was irregular and the planes of contact we re rarely flat or simple. Variability in cell shape was indicated by the ra nge in surface area to volume ratios, from 0.080 to 0.198 mu m(-1). Structu ral detail at the internal surface of the cell wall was apparent. The techn ique is applicable to a wide range of morphometric analyses in plant cell b iology, particularly developmental studies, and reveals details of cell siz e and shape that were previously unattainable.