  Single-molecule narrowfield microscopy is a versatile tool to investigate a
diverse range of protein dynamics in live cells and has been extensively used
in bacteria. Here, we describe how these methods can be extended to larger
eukaryotic, yeast cells, which contain sub-cellular compartments. We describe
how to obtain single-molecule microscopy data but also how to analyse these
data to track and obtain the stoichiometry of molecular complexes diffusing in
the cell. We chose glucose mediated signal transduction of live yeast cells as
the system to demonstrate these single-molecule techniques as transcriptional
regulation is fundamentally a single molecule problem - a single repressor
protein binding a single binding site in the genome can dramatically alter
behaviour at the whole cell and population level.
