A real-time view of life within 100 nm of the plasma membrane

Key Points Evanescent field (EF) fluorescence microscopy (also called total internal reflection fluorescence microscopy) is uniquely suited to image the plasma membrane with its associated organelles and macromolecules in living cells Total internal reflection of a light beam generates an evanescent field ? a thin layer of light that typically penetrates about 40?200 nm from a coverslip into an adhering cell. EF fluorescence microscopy combines the specificity of confocal microscopy to detect fluorescent molecules with a depth discrimination of near-molecular dimensions. However, imaging is confined to the cell surface. A recent wave of applications was aimed at membrane transport events in living cells. EF fluorescence microscopy offers new insights for its abilities to resolve even the smallest vesicles made by cells and to detect molecular-sized motions of fluorescent objects vertical to the glass. In endocrine cells and synaptic nerve terminals, transport, exocytosis and replenishment of single secretory vesicles could be investigated. No method existed to record signals from single synaptic vesicles before the exocytic event. EF fluorescence imaging of transport containers undergoing constitutive exocytosis provided a wealth of new results, including evidence for incomplete exocytosis. Other studies enhanced our understanding about the role of actin filaments for the movement of small organelles near the plasma membrane. An important use of EF fluorescence microscopy will be to image signal-transduction events. Recent studies using EF fluorescence microscopy have shown that biochemistry can be done in living cells at the level of single molecules. The plasma membrane is a two-dimensional compartment that relays most biological signals sent or received by a cell. Signalling involves membrane receptors and their associated enzyme cascades as well as organelles such as exocytic and endocytic vesicles. Advances in light microscope design, new organelle-specific vital stains and fluorescent proteins have renewed the interest in evanescent field fluorescence microscopy, a method uniquely suited to image the plasma membrane with its associated organelles and macromolecules in living cells. The method shows even the smallest vesicles made by cells, and can image the dynamics of single protein molecules.