Multiparameter evanescent-wave imaging in biological fluorescence microscopy

Wide-field optical microscopy of live cells with nearfield precision, tracking the dynamics of subcellular organelles, imaging of single-molecule reactions with millisecond time-resolution, or watching synaptic nerve terminals "in action"-these are some examples of recent work that triggered the "renaissance" of evanescent-wave (EW) spectroscopy in biological imaging. In these studies, the goal is to markedly reduce background fluorescence from locations in the sample other than the cell/substrate interface. After a brief reminder of EW generation, we discuss how the confinement of fluorescence excitation highlights cellular structure near the plasma membrane with unprecedented detail. We then illustrate how the intensity distribution and polarization of the EW can be used to study dynamic biological processes that have neither been accessible with optical (confocal or two-photon fluorescence) nor electron microscopy, and take a glimpse of what is to come in EW imaging.