Optical subsectioning and multiple focal-plane imaging in the standing-wave fluorescence microscope

Standing-wave fluorescence microscopy (SWFM) is a direct-imaging method through which very high axial resolution can be obtained in certain types of biological specimens. In the microscope, interference of light is used to produce a standing-wave field through the specimen, so that it is illuminated in a pattern of planar zones, rather than uniformly. Fluorescence is excited with spatial selectivity in proportion to the intensity pattern. In the instruments in use at present, two nearlycollimated s-polarized beams from a laser are directed into the specimen from opposite sides at mirrorimage angles with respect to the microscope axis (Figure). The alternating nodal and antinodal planes of the field are in this case parallel to the object focal plane, with a node spacing equal to λo/(2n cos θ), where θ is the beam angle, and n is the refractive index of the specimen. The minimum node spacing, λo/2n, is obtained when the beams are counterpropagating on-axis, and is typically 0.18 μm. In the simplest SWFM system, a mirror is placed behind the specimen such that an on-axis beam interferes with its reflection. In our dual-beam instrument, the laser output is split, and the beams directed into the specimen through a pair of opposed high-NA objectives. In both cases, the standing-wave field planes can be shifted axially through the specimen, by control of the phase of one of the beams. This can be done with high precision by use of a piezoelectric mirror drive. For SWFM to work well, it is necessary that the specimen be a weak phase object, i.e., that the refractive index heterogeneity be small.