Confocal Microscopy: Recent Developments

Abstract

Publisher Summary Microscopy, and in particular confocal microscopy and two-photon excitation, provide a three-dimensional resolution; therefore, a three-dimensional resolution criterion is necessary that documents that a resolution improvement along one axis is not imposed at the expense of a decrease along another axis. Some techniques using—for example, annular apertures—improve the lateral resolution but at the same time degrade the axial resolution. The confocal microscope reduces the illumination field and the detection field to the physical limit determined by diffraction and hence discriminates all light emitted outside the focal volume. In a 4Pi(A) confocal microscope, two opposing lenses use spherical waves to illuminate a focal spot coherently and produce a standing wave that modulates the intensity along the optical axis. Two minima are slightly more than λ/2n apart. By adjusting the phase and moving the object relative to the focal spot while recording the fluorescence intensity as a function of the position, images are generated. In the confocal theta microscope, the axial resolution can be improved by a factor of three, and low-NA systems may have axial resolutions that are better than those achieved with confocal high-NA systems.