The use of fluorescence microscopy for investigating the three-dimensional structure of cells and tissue is of growing importance in cell biology, biophysics and biomedicine. Three-dimensional data are obtained by recording a series of images of the specimen as it is stepped through the focal plane of the microscope. Whether by direct imaging or by confocal scanning, diffraction effects and noise generally limit axial resolution to about 0.5 μm. Here we describe a fluorescence microscope in which axial resolution is increased to better than 0.05 μm by using the principle of standing-wave excitation of fluorescence. Standing waves formed by interference in laser illumination create an excitation field with closely spaced nodes and antinodes, allowing optical sectioning of the specimen at very high resolution. We use this technique to obtain images of actin fibers and filaments in fixed cells, actin single filaments in vitro and myosin II in a living cell.