Enhanced axial resolution of the cytoskeleton in fluorescence microscopy by standing-wave excitation

Abstract

When the depth-of-field of a microscope is less than the axial dimension of the specimen, 3d information can be derived from a set of images recorded as the specimen is stepped through the object focal plane of the microscope. This procedure, known as optical sectioning microscopy (OSM), is the same in direct imaging and confocal scanning. For both of these cases in fluorescence microscopy, axial (depth) resolution is more limited than transverse resolution, for fundamental reasons. Our research aim has been to enhance axial resolution in fluorescence OSM (FOSM) while retaining the high-speed information transfer characteristics of direct imaging that are necessary for 3d studies of living cells in culture.Standing-wave fluorescence microscopy (SWFM) is a direct imaging method in which the object is illuminated by a three-dimensional field of planar interference fringes (standing waves) oriented parallel to the focal plane of the microscope. This field is produced in the specimen by crossing two coherent, collimated, s-polarized beams of equal amplitude directed through the specimen at complementary angles (θ, π -θ) relative to the axis of the microscope.