Three-dimensional super-resolution with a 4Pi-confocal microscope using image restoration

The combination of two-photon excitation 4Pi-confocal fluorescence microscopy with image restoration leads to a fundamental improvement of three-dimensional resolution in the imaging of transparent, fluorescent specimens. The improvement is exemplified by randomly dispersed fluorescent beads and with actin filaments in a mouse fibroblast cell. For an illumination wavelength of 810 nm, we obtained lateral and axial full-width at half-maxima of point-like objects of 120-140 nm, and 70-100 nm, respectively. Fluorescent beads that are 150 nm apart are imaged with an intensity dip of similar to 25%. This amounts to a improvement of the axial resolution over;sixfold standard two-photon confocal microscopy. In the cell, the 3D-images reveal details otherwise not resolvable with focused light. (C) 1998 American Institute of Physics. [S0021-8979(98)00920-7].