## 3 Results
### High-Resolution 3D Imaging Improves Cell Distinction but Greatly Reduces the Field of View  
Using high-resolution settings, the observed field of view was restricted. However, a much larger number of details can be distinguished (Figure 5A,B). In order to facilitate the interpretation of the images, light microscopy images of wheat grains at similar development stages, stained with toluidine blue O to highlight cell structures, are presented  
Figure 4: Virtual slices within low-resolution images of whole grains at contrasted development stages. (**A**) XY slice at middle \(z\)-position for grain at 100 \({}^{\circ}\)DAA. (**B**,**C**) XZ slice at middle \(y\)-position and YZ slice at middle \(x\)-position of the same grain. (**D**) XY slice in the bottom of the grain, showing a portion of the embryo. (**E**–**H**) same representations for a grain at 250 \({}^{\circ}\)DAA. Scale bar represents 1000 \(\upmu\)m.  
(Figure 5C,D). Differences between tomography and microscopy images may be due to the differences between grains, but also to preparation artifacts in microscopy images. On the tomography images, individual cells are clearly distinguished in several tissues. In the pericarp parenchyma, cells are surrounded by intercellular spaces and are easily distinguished. Cell layers of the inner pericarp (cross cells and tube cells) can be recognized between the endosperm and the pericarp parenchyma (Figure 6A,C). Individual cells within tissues with no voids are also detected due to differences in contrast which reflect differences in density/composition. This is the case for cells of the nucellar epidermis, nucellar projection, and modified aleurone at 250 \({}^{\circ}\)DAA for which cell walls appear thick and bright while their cytoplasm appears dark (Figures 5B and 6A,B). The contours of some cells in the inner compartment can be seen at 100 \({}^{\circ}\)DAA but these cells are not as recognizable as at 250 \({}^{\circ}\)DAA. At 250 \({}^{\circ}\)DAA, almost all tissue layers revealed by optical microscopy were observed using high-resolution tomography. Cells surrounding the vascular bundle were less defined and the testa was not revealed by the X-rays (Figure 6B). The testa lays between the nucellar epidermis and the inner pericarp which are both distinguished. The main difference between the two techniques is that the inside of cells is not contrasted in the X-ray images while it is well-defined using histological staining and microscopy. The only detail  
Figure 5: High-resolution images of grain tissues. (**A,B**) XY slices of 3D tomography images of a wheat grain at 100 \({}^{\circ}\)DAA and 250 \({}^{\circ}\)DAA in the crease region. (**C,D**) Microscopy images of wheat grain at 100 \({}^{\circ}\)DAA and 250 \({}^{\circ}\)DAA (Toluidine Blue O stained) in similar regions as (**A,B**). of the inside of cells revealed by X-rays is the presence of starch grains in the endosperm that are particularly highlighted due to their high density. The endosperm cavity, which contains water, proteins, and carbohydrates, is visible in the ventral crease region although its shape appears different than in microscopy images of grain sections where it is altered and emptied by preparation steps (Figure 5B,D) [21]. At 100 \({}^{\circ}\)DAA (Figure 5A), apart from pericarp tissues, fewer internal tissues are distinguished in the X-ray images probably because their developing cell walls are thinner and because the wheat grains are more highly hydrated (75% and 68% at 100 \({}^{\circ}\)DAA and 250 \({}^{\circ}\)DAA respectively). Microscopy imaging revealed a large number of cells (Figure 5C,D and Figure 6C,D), delineated by their cell wall. The coloration of the cell walls also provided information about their chemical composition. With our tomography setups cell walls were visible only in some tissues (e.g., nucellar epidermis, modified aleurone), making most cells visible only within highly porous tissues.