The attachment of bacteria to host roots is an initial step in colonization and is influenced by several factors including bacterial transport, surface recognition, and local shear forces. A quantitative understanding of the affinity of proteobacteria for root surfaces and a clear picture of the initial adhesion kinetics associated with attachment is essential to characterize biofilm formation and root colonization. The dual function of imaging and measuring interaction forces makes Atomic Force Microscopy (AFM) a unique tool for studying bacterial adhesion. The adhesion between the cell surface and the AFM tip can be measured from the extension and the retraction of the force curve cycle using functionalized cantilever tips. The mechanisms involved in the bacterial attachment and motility behavior were elucidated by force spectroscopy measurements and microfluidic systems. The developed analytical techniques quantified the adhesion forces which helped to explain the spatial and temporal dynamics of the colonization of Populus by proteobacteria. The role of the extracellular polymeric substance and the pili in facilitating the bacterial adhesion during biofilm formation are being analyzed.