<b>4Pi‐confocal images with axial superresolution</b>

Abstract

We present two‐photon excitation 4Pi‐confocal images of clustered fluorescence beads demonstrating three‐dimensional far‐field light microscopy with unprecedented resolution. For an excitation wavelength of 760 nm, the lateral and axial resolution amounts to 200 and 145 nm, respectively. The four‐fold improved axial resolution is achieved by engineering the point‐spread function through a suitable combination of aperture enlargement, two‐photon excitation, confocalization and three‐point deconvolution. In contrast to their confocal counterparts, 4Pi‐confocal images do not exhibit the typical axial elongation. The axial resolution in the 4Pi‐confocal images corresponds to about one‐fifth of the wavelength and surpasses the lateral resolution by 25%.