X-ray microscopy enables multiscale high-resolution 3D imaging of plant cells, tissues, and organs

Capturing complete internal anatomies of plant organs and tissues within their relevant morphological context remains a key challenge in plant science. While plant growth and development are inherently multiscale, conventional light, fluorescence, and electron microscopy platforms are typically limited to imaging of plant microstructure from small flat samples that lack direct spatial context to, and represent only a small portion of, the relevant plant macrostructures. We demonstrate technical advances with a lab-based X-ray microscope (XRM) that bridge the imaging gap by providing multiscale high-resolution 3D volumes of intact plant samples from the cell to whole plant level. Serial imaging of a single sample is shown to provide sub-micron 3D volumes co-registered with lower magnification scans for explicit contextual reference. High quality 3D volume data from our enhanced methods facilitate more sophisticated and effective computational segmentation and analyses than have previously been employed for X-ray based imaging. Advances in sample preparation make multimodal correlative imaging workflows possible, where a single resin-embedded plant sample is scanned via XRM to generate a 3D cell-level map, and then used to identify and zoom in on sub-cellular regions of interest for high resolution scanning electron microscopy. In total, we present the methodologies for use of XRM in the multiscale and multimodal analysis of 3D plant features using numerous economically and scientifically important plant systems.