  Roughly half of a cells proteins are located at or near the plasma membrane.
In this restricted space the cell senses its environment, signals to its
neighbors and ex-changes cargo through exo- and endocytotic mechanisms. Ligands
bind to receptors, ions flow across channel pores, and transmitters and
metabolites are transported against con-centration gradients. Receptors, ion
channels, pumps and transporters are the molecular substrates of these
biological processes and they constitute important targets for drug discovery.
Total internal reflection fluorescence microscopy suppresses background from
cell deeper layers and provides contrast for selectively imaging dynamic
processes near the basal membrane of live-cells. The optical sectioning of
total internal reflection fluorescence is based on the excitation confinement
of the evanescent wave generated at the glass-cell interface. How deep the
excitation light actually penetrates the sample is difficult to know, making
the quantitative interpretation of total internal reflection fluorescence data
problematic. Nevertheless, many applications like super-resolution microscopy,
colocalization, fluorescence recovery after photobleaching, near-membrane
fluorescence recovery after photobleaching, uncaging or
photo-activation-switching, as well as single-particle tracking require the
quantitative interpretation of evanescent-wave excited images. Here, we review
existing techniques for characterizing evanescent fields and we provide a
roadmap for comparing total internal reflection fluorescence data across
images, experiments, and laboratories.
