3-DIMENSIONAL SUPERRESOLUTION WITH A 4PI-CONFOCAL MICROSCOPE USING IMAGE-RESTORATION

The combination of two-photon excitation 4Pi-confocal fluorescence mic roscopy with image restoration leads to a fundamental improvement of t hree-dimensional resolution in the imaging of transparent, fluorescent specimens. The improvement is exemplified by randomly dispersed fluor escent beads and with actin filaments in a mouse fibroblast cell. For an illumination wavelength of 810 nm, we obtained lateral and axial fu ll-width at half-maxima of point-like objects of 120-140 nm, and 70-10 0 nm, respectively. Fluorescent beads that are 150 nm apart are imaged with an intensity dip of similar to 25%. This amounts to a improvemen t of the axial resolution over;sixfold standard two-photon confocal mi croscopy. In the cell, the 3D-images reveal details otherwise not reso lvable with focused light. (C) 1998 American Institute of Physics. [S0 021-8979(98)00920-7].