Correlative microscopy for in-depth analysis of calcium oxalate crystals in plant tissues

Calcium oxalate (CaOx) crystals are commonly found in many plant species. These crystals vary in distribution and morphology and to elucidate their role in plants multiple methods have been applied. Raman imaging and polarized light microscopy (PLM) easily visualize the crystals within plant tissues, but both methods are limited in spatial resolution by the diffraction of light. To unravel the distinctive shape and morphology of CaOx crystals down to the nanoscale and how they are embedded within cells, high resolution scanning electron microscopy is needed. To grasp the full potential of multiple methods in CaOx studies, a novel and easy-to-build correlative sampling approach is presented on different nut species (pecan (Carya illinoinensis), Turkish hazel (Corylus colurna) and black walnut (Juglans nigra)), including soft tissues (young developmental stages) as well as hard tissues (mature nutshells).

Young seed coat tissues as well as mature nutshells included distinct morphological CaOx features, like druses and prismatic crystals. By Raman imaging the chemical composition of all investigated crystals was verified as calcium oxalate monohydrate (COM) and Raman band intensity changed according to crystal plane orientation with respect to incident laser polarisation. Calcium oxalate dihydrate (COD) was only found in the young C. illinoinensis seed coat and was restricted to a few pixels adjacent to cell walls. These thin cell walls were identified as pectin-rich, while in the mature nutshells the crystals were surrounded by thicker and highly lignified cell walls. The Raman and light microscopy results were correlated with SEM images, which gave additional information on crystal surface structure and/or internal porosity on the nanoscale.

The presented correlative approach preserved the structural integrity of crystals and cellular structures during cutting and transferring between microscopes. Analysing exactly the same sample (position) by Raman, polarized light microscopy and SEM opens the view on the distribution within tissues and cells as well as the molecular structure of the crystals and adjacent cell structures. Such a comprehensive in-situ characterization paves the way for a better understanding of mineralization processes of different minerals in all kinds of biological tissues.