Nanoscopic Cellular Imaging: Confinement Broadens Understanding

In recent years, single-molecule fluorescence imaging has been reconciling a fundamental mismatch between optical microscopy and subcellular biophysics. However, the next step in nanoscale imaging in living cells can be accessed only by optical excitation confinement geometries. Here, we review three methods of confinement that can enable nanoscale imaging in living cells: excitation confinement by laser illumination with beam shaping; physical confinement by micron-scale geometries in bacterial cells; and nanoscale confinement by nanophotonics.