<p>Integral membrane proteins are crucial molecules ubiquitous to all cell types, coordinating cell signalling and facilitating the tightly regulated transport of essential nutrients across plasma membrane. Defects in membrane proteins are associated with disease, emphasising the need to understand the structural, mechanistic and regulatory mechanisms which control integral membrane proteins. Recent technological advances in optical microscopy have allowed appropriate study of these small proteins using tools with molecular resolution which can non-invasively observe their native organisation in the plasma membrane in situ<i>.</i> Complimentarily, by utilising photochemical phenomena and analyses, single-molecule detail can be elucidated from conventional microscope systems. In this review, we firstly overview the methodologies used for studies of membrane proteins and then review the biophysical results gleaned from their application with an emphasis on membrane transporters. We show that single molecule studies of integral membrane proteins are beginning to unveil striking new regulatory mechanisms with wide applicability across many distinct fields of biological research.</p>