AbstractFor fundamental reasons, fluorescence microscopes are more limited in axial, as opposed to transverse, resolution. By giving the excitation field a particular axial structure, this limitation can be partially alleviated, as in confocal scanning or two‐photon scanning, or even in optical sectioning microscopy in cases where the object occupies only a small part of the field of view. Standing‐wave fluorescence microscopy (SWFM) is a direct imaging method in which the specimen is excited by a 3‐D field of planar interference fringes oriented parallel to the object focal plane of the microscope. By shifting the position of the nodal planes of this field relative to the specimen, structures that are normally obscured under uniform excitation become resolved. We demonstrate that, in very thin biological specimens, thisoptical subsectioningincreases axial resolution by an order of magnitude, to 0.04 μm. In comparison to confocal scanning, SWFM resolves fine axial structure with more than 10‐fold greater speed, and with similarly‐reduced photobleaching. We also discuss the more general case of excitation field synthesis (EFS), in which standing wave fields differing in node spacing can be overlapped or time‐multiplexed in the specimen so that the average field is non‐periodic and peaked at the object focal plane. A transfer function model is given to show that for weakly‐refractive specimens of arbitrary thickness, such as single cells or cell monolayers, EFS should lead to a fivefold improvement in