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.
Ibáñez-López
Cristina
C
Applied Optics Laboratory, ENSTA-Ecole Polytechnique, 91761 Palaiseau, France.
Saavedra
Genaro
G
Plamann
Karsten
K
Boyer
Gilbert
G
Martínez-Corral
Manuel
M
D000465:Algorithms; D007091:Image Processing, Computer-Assisted; D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation