The ultimate goal of cell biology is to understand how proteins function in real cells and to understand their regulatory mechanisms. To achieve this goal one needs to analyze molecules in their cellular environment and not under idealized test tube conditions. A major difference between in vivo and in vitro conditions is the crowdedness (and associated molecular complexity) of the cytoplasm and biological membranes. As a consequence diffusion in this environment is significantly slower. To make biology more quantitative, we should measure the key parameters such as reaction rates and diffusion coefficients in vivo. Here, I propose an experimental set-up to measure the diffusion of membrane proteins in live bacterial cells. The main objective of this project is to establish a relationship between the diffusion coefficient and number of trans-membrane helices (radius in the membrane) of membrane proteins. The mobility of fluorescently labeled proteins will be probed at the single molecule level in the membranes of a model prokaryotic organism – Escherichia coli. Single molecule tracking of membrane proteins systematically increasing in size (radius in the membrane) will be implemented to obtain a general model of protein diffusion in the membranes. Assigning actual numbers to the parameters of a cell will provide a quantitative context for systems biology efforts and sharpen our understanding of how a cell works. By training-through-research, the fellowship will allow me to learn new techniques (single molecule tracking, programming) and how to build/align microscope set-ups. This will help me transform from a microscope user to a microscope constructor and vastly increase my potential as a cell biologist. In the international and interdisciplinary environment of the host group I will improve my ability to train and manage people, learn to translate my science, across disciplines, to a new audience and to translate fundamental research into industrial applications.