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 observa tion are tilted by an angle V. One lens is used to illuminate the samp le and one lens is used to observe the light emitted by the sample. In a confocal theta arrangement the axial discrimination is substantiall y improved over a linear confocal arrangement and the observation volu me 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 poi nt spread function is decreased by a factor of 2.2. This means that th e point spread function of a confocal theta two-photon microscope oper ating at a wavelength of 780 nm has an axial full width half maximum o f 230 nm. In a 4Pi(A)-confocal theta microscope the axial side lobes a re removed.