Abstract
Computed tomography remains strongly underused in plant sciences despite its high potential in delivering detailed 3D phenotypical information because of the low X-ray absorption of most plant tissues. Existing protocols to study soft tissues display poor performance, especially when compared to those used on animals. More efficient protocols to study plant material are therefore needed. Flowers of Arabidopsis thaliana and Marcgravia caudata were immersed in a selection of contrasting agents used to treat samples for transmission electron microscopy. Grayscale values for floral tissues and background were measured as a function of time. Contrast was quantified via a contrast index. The thick buds of Marcgravia were scanned to determine which contrasting agents best penetrate thick tissues. The highest contrast increase with cytoplasm-rich tissues was obtained with phosphotungstate, whereas osmium tetroxide and bismuth tatrate displayed the highest contrast increase with vacuolated tissues. Phosphotungstate also displayed the best sample penetration. Furthermore, infiltration with phosphotungstate allowed imaging of all plants parts at a high resolution of 3 µm, which approaches the maximum resolution of our equipment: 1.5 µm. The high affinity of phosphotungstate for vasculature, cytoplasm-rich tissue, and pollen causes these tissues to absorb more X-rays than the surrounding tissues, which, in turn, makes these tissues appear brighter on the scan data. Tissues with different brightness can then be virtually dissected from each other by selecting the bracket of grayscale to be visualized. Promising directions for the future include in silico phenotyping and developmental studies of plant inner parts (e.g., ovules, vasculature, pollen, and cell nuclei) via virtual dissection as well as correlations of quantitative phenotypes with omics datasets. Therefore, this work represents a crucial improvement of previous methods, allowing new directions of research to be undertaken in areas ranging from morphology to systems biology.
Highlights
The phenotype, i.e. the total sum of the observable characteristics of an organism, needs to be quantified both with precision and detail in order to harvest the full potentialities of the omics era
In order to demonstrate the versatility of our methods, larger and more complex flowers were scanned from two other species: Calycanthus floridus (Calycanthaceae), and Haplophyllum lissonotum (Rutaceae)
Arabidopsis thaliana samples and buds of Marcgravia caudata were fixed in FAA (50 vol ethanol 100%, 5 vol glacial acetic acid, 10 vol formaldehyde 37%, 35 vol dH2O) and washed with 70% ethanol (EtOH) prior to infiltration
Summary
The phenotype, i.e. the total sum of the observable characteristics of an organism, needs to be quantified both with precision and detail in order to harvest the full potentialities of the omics era. In order obtain high resolution and high magnification imaging, the protocols for plant tissues involve critical point drying of the samples [36,37] and infiltration with iodine [36] or toxic uranium acetate [37]. These protocols [36,37] do not include quantification of contrast increase nor proved satisfactory when replicated, because they caused only weak contrast increase and displayed poor tissue penetration.
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