Plant tissues in 3D via X-Ray tomography: methods towards high resolution imaging and applications

Autor(en)

Yannick Städler, Jürg Schönenberger, David Masson

Abstrakt

Study of plant morphology, development, function, and ultimately evolution, requires detailed three-dimensional visualization and modelling. X-ray microtomography (micro-CT) allows straightforward visualization and modelling of complex plant and animal samples. However, the low absorbance of most plant tissues has earlier been an obstacle for the use of this method in plant sciences (with the notable exception of wood and permineralized, or charcoalified fossils). Here we present staining methods that allow for high contrast and high resolution reconstructions of any plant tissue/organ using commercial MicroCT systems. In addition, we present a series of applications that we developed in our lab. Stains were selected from the transmission electron microscopy literature. Fixed Arabidopsis thaliana flowers were infiltrated for 12h to a week. We quantified penetration of the tissue, contrast improvement, selectivity and homogeneity of the stain. Phosphotungstate proved to be the most efficient non-destructive stain for tissue penetration and contrast increase, which occurs in a homogeneous but selective way (with a stronger increase for cytoplasm-rich tissues). We developed special protocols to reduce shrinkage and to obtain maximal resolution (3µm) on small samples. Staining methods, by allowing straightforward imaging of fixed material, open a broad range of possible applications. Micro-CT can be used to study samples that are difficult to study via serial sectioning or electron microscopy. It can also be applied to the study of quantitative traits that can be regressed against ‘omics’ datasets, e.g. metabolomic data as in our study of the developmental trajectory of Arabidopsis flowers. Moreover, it can also be applied to the study of pollination, where precise 3d matches between pollinator and flower morphologies can be quantified, e.g. in our study of the morphological covariation between the sexually deceptive orchid Ophrys, its pollinators (male bees), and what the orchid mimics (female bees). Phosphotungstate increases the contrast of some tissues more than others (e.g. pollen vs. parenchyme), therefore by selecting a bracket of greyscale to be displayed, it is possible to separate different components from each other, thereby virtually dissecting the flowers. Once a tissue is dissected from the rest of the flower, volume information is readily available, allowing e.g. to count large numbers of pollen grains with a small errors (ca. 2%). This method may be used to quantify pollen to ovule ratios in plant for which this information is difficult to gather (Orchidaceae), in a much more straightforward, and more reliable way than traditional counting techniques.

Organisation(en)

Externe Organisation(en)

Federal Office of Meteorology and Climatology, MeteoSwiss

Publikationsdatum

2013

ÖFOS 2012

106042 Systematische Botanik, 106012 Evolutionsforschung, 106008 Botanik

Link zum Portal

https://ucris.univie.ac.at/portal/de/publications/plant-tissues-in-3d-via-xray-tomography-methods-towards-high-resolution-imaging-and-applications(4fd6bd4a-cca8-4996-8809-926801ff139c).html