Quasi‐spherical focal spot in two‐photon scanning microscopy by three‐ring apodization

We present a beam‐shaping technique for two‐photon excitation (TPE) fluorescence microscopy. We show that by inserting a properly designed three‐ring pupil filter in the illumination beam of the microscope, the effective optical sectioning capacity of such a system improves so that the point spread function gets a quasi‐spherical shape. Such an improvement, which allows the acquisition of 3D images with isotropic quality, is obtained at the expense of only a small increase of the overall energy in the axial sidelobes. The performance of this technique is illustrated with a scanning TPE microscopy experiment in which the image of small beads is obtained. We demonstrate an effective narrowing of 12.5% in the axial extent of the point spread function, while keeping the 82% of the spot‐fluorescence efficiency. Microsc. Res. Tech. 67:22–26, 2005. © 2005 Wiley‐Liss, Inc.