Biological processes that occur in the soil have important environmental implications. These processes include root growth and microbial interactions with roots and so il particles and they influence the efficiency of crop production and, in turn, global food security. The observation and imaging of these below-ground processes is difficult due to the opacity of soil and so this thesis presents a new artificial soil analogue that is transparent and therefore allows optical imaging. Transparent soil is a 3D matrix of chemically treated particles of the low refractive index fluoropolymer Nafion, water, plant nutrients and air and has water retention and ion exchange properties similar to natural soils. Before imaging, the transparent soil was saturated with a refractive index matched liquid for appropriate transparency. The substrate was used for 3D imaging of living root systems and high resolution imaging of living roots at a cellular level in relation to the fluorescent-labelled Nafion particles of the substrate. Soil physical conditions influence the growth rate and direction of roots. The substrate compaction and particle size range was varied in transparent soil to quantify the effect of these conditions on 3D root trajectories of lettuce plants. Root systems of plants grown in different substrate conditions were imaged and the root lengths were measured along with the curvature and verticality at sequential points along the roots. There was a greater range of root curvatures in substrates with larger particl e size s and deviation from vertical increased with distance along the root . In substrates with different compactions, there was no effect of compaction on the root curvature or verticality measurements, however the measurements were influenced by the distance along the root. Soil microbes were also studied using the transparent soil system. Pseudomonas fluorescens, a plant growth promoting rhizobacteria, associates closely with plant roots and can act as a biocontrol agent by conveying pathogen resistance to the plant. For this reason, the interaction between lettuce roots and GFP labelled P. fluorescens was studied with the aim of quantifying colonisation patterns along the root and the abundance of bacteria in the substrate surrounding the roots. Transparent soil with two different particle size categories was used to investigate if the substrate particle size affected the colonisation of the roots. Imaging of living roots and bacteria was carried out at 3D sample points along the root and adjacent to the root and it was found that there was a greater abundance of bacteria on the roots than in the substrate. There was a consistent base level of bacterial fluorescence in imaging points that did not include roots, regardless of whether or not there was a plant in the sample and the distance from the root. Substrate particle size had no effect on root colonisation.