FUNDAMENTAL REDUCTION OF THE OBSERVATION VOLUME IN FAR-FIELD LIGHT-MICROSCOPY BY DETECTION ORTHOGONAL TO THE ILLUMINATION AXIS - CONFOCAL THETA MICROSCOPY

In a theta microscope the axis of illumination and the axis of observation are tilted by an angle V. One lens is used to illuminate the sample and one lens is used to observe the light emitted by the sample. In a confocal theta arrangement the axial discrimination is substantially improved over a linear confocal arrangement and the observation volume becomes spherical. Using a numerical aperture of 0.94 in water the axial resolution is improved by a factor 3.5 and the volume of the point spread function is decreased by a factor of 2.2. This means that the point spread function of a confocal theta two-photon microscope operating at a wavelength of 780 nm has an axial full width half maximum of 230 nm. In a 4Pi(A)-confocal theta microscope the axial side lobes are removed.