Enhancement in statistical and image analysis for in situ µSXRF studies of elemental distribution and co-localization, using Dioscorea balcanica

Synchrotron-radiation-based X-ray microfluorescence has been used for in situ investigation of the distribution of micronutrient and macronutrient elements in an unstained cross section of a stem of monocotyledonous liana plant Dioscorea balcanica Košanin. The elemental allocation has been quantified and the grouping/co-localization in straight and twisted stem internodes has been analysed. Synchrotron-based X-ray microfluorescence (µSXRF) is an analytical method suitable for in situ investigation of the distribution of micronutrient and macronutrient elements in several-micrometres-thick unstained biological samples, e.g. single cells and tissues. Elements are mapped and quantified at sub-p.p.m. concentrations. In this study the quantity, distribution and grouping/co-localization of various elements have been identified in straight and twisted internodes of the stems of the monocotyledonous climber D. balcanica Košanin. Three different statistical methods were employed to analyse the macro-nutrient and micronutrient distributions and co-localization. Macronutrient elements (K, P, Ca, Cl) are distributed homogeneously in both straight and twisted internodes. Micronutrient elements are mostly grouped in the vasculature and in the sclerenchyma cell layer. In addition, co-localization of micronutrient elements is much more prominent in twisted than in straight internodes. These image analyses and statistical methods provided very similar outcomes and could be applied to various types of biological samples imaged by µSXRF