Sharp Spherical Focal Spot by Dark Ring 4Pi-Confocal Microscopy

A major goal of improving the resolution in far-field light microscopy is to create observation volumes that are sharp and spherical. Two-photon excitation 4Pi-confocal fluorescence microscopy provides a sharp and nearly spherical focal diffraction maximum, but the maximum is accompanied by axial side-lobes of 30-40% relative height. Whereas in 4Pi-confocal imaging the effect of the lobes on the image can be eliminated by data processing, in spectroscopy their presence contributes to the observation volume in full. Here we show that simple binary amplitude filters, placed in the entrance aperture of novel 1.45 numerical aperture oil immersion lenses, suppress the side-lobes below 7%. As a result, a nearly spherical sharp observation volume is created consisting of a solitary spot of similar to150 nm full-width-half-maximum.