Technical Advance: 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 digital image reconstruction. The whole berry was fixed, bisected longitudinally, cleared in methyl salicylate, stained with safranin O and mounted in methyl salicylate. Optical slices were collected at 1.0 µm intervals to a depth of 150 µm. Neighbouring z-series were joined post-collection to double the field-of-view. Attenuation at depth of the fluorescent signal from cell walls was quantified and corrected. Axial distortion due to refractive index mismatch between the immersion and mounting media was calibrated using yellow-green fluorescent microspheres and corrected. Transmission electron microscopy was used to correct fluorescent measurements of cell wall thickness. Digital image reconstructions of wall-enclosed spaces enabled cells to be rendered as geometric solids of measurable surface area and volume. Cell volumes within the inner mesocarp tissue of a single grape berry exhibited a 14-fold range, with polysigmoidal distribution and groupings around specific size classes. Cell shape was irregular and the planes of contact were rarely flat or simple. Variability in cell shape was indicated by the range in surface area to volume ratios, from 0.080 to 0.198 µm-1. Structural detail at the internal surface of the cell wall was apparent. The technique is applicable to a wide range of morphometric analyses in plant cell biology, particularly developmental studies, and reveals details of cell size and shape that were previously unattainable.