Calibrating evanescent-wave penetration depths for biological TIRF microscopy

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.