Two New High-Resolution Confocal Fluorescence Microscopies (4Pi, Theta) with One- and Two-Photon Excitation

Abstract

Improving spatial resolution has been one of the main goals of research since the early beginnings of light microscopy. As a microscope objective lens cannot cover more than 35% of the full solid angle of 4rc steradians, the intensity distribution of a focused spot is elongated along the optical axis and the axial resolution in a conventional microscope is generally many times poorer than the lateral resolution. Much effort has been exerted to reduce the axial extent of this light distribution. An important step toward an improved axial resolution was the development of confocal arrangements (Minsky, 1961; Brakenhoff et al., 1979; Wilson et al., 1980; Wijnaendts van Resandt et al., 1985; Carlsson et al., 1985) that permit the investigation of thick samples along their optical axis. However, the axial resolution in a confocal microscope is still poorer than the lateral resolution (Wilson and Sheppard, 1984, pp. 70–72). Further improvement was limited by the fact that, according to diffraction theory, an improvement is only feasible by decreasing the wavelength or by increasing the numerical aperture (NA) of the objective lens.