Abstract3D imaging techniques, such as serial block-face scanning electron microscopy (SBF-SEM) and X-ray microscopy (XRM), have increasingly provided critical insights into complex and multi-dimensional biological structures, including plants. However, plant specimens face many challenges when processing for microscopy due to their hydrophobic cuticle, chemically diverse cell walls and intercellular air spaces that can impede adequate chemical fixation and staining. Since SBF-SEM and XRM depend on high atomic number contrast agents for improved throughput, resolution and image contrast, we optimized a heavy metal staining protocol to allow direct correlation of the same specimen with both techniques. Tobacco leaf tissues were fixed in paraformadehyde and glutaraldehyde followed by osmium tetroxide and then stained with potassium ferrocyanide, osmium-thiocarbohydrazide-osmium (OTO), hot uranium and lead salts and embedded in Quetol resin. Samples were imaged on ZEISS Xradia 520 and 620 Versas and then a selected area was targeted and 2000+ consecutive SBF-SEM images acquired on a ZEISS GeminiSEM 300 equipped with a Gatan 3View. We applied ORS Dragonfly for 3D volume correlation and deep learning for improved segmentation of XRM and SBF-SEM. With this approach, we were able to discern and segment chloroplasts, starch, nuclei and cell walls within XRM volumes. Additionally, we were able to readily resolve and contrast many membrane features including the spacing within grana stacks. Our results demonstrated the utility of increased metalization for high-resolution 3D volume imaging of key plant structures and multiscale correlative XRM to EM workflows of plants.
Dr Ruth Chalmers-Redman
Global Business Development - Life Science Electron Microscopy, Cryo, Bio XRM - ZEISS Research Microscopy Solutions
Dr Redman holds a Neuroscience PhD and has worked with Zeiss for 16 years having performed roles from confocal specialist to electron, helium ion, x-ray microscopy specialist for the life sciences. She has led a team of specialists in the Asia Pacific region and most recently, Ruth performs globally with a focus on life science cryo SEM, multiSEM and X-ray microscopy business and application development.