Multispot point spread function for multiphoton fluorescence microscopy

We propose and demonstrate an imaging technique capable of generating multiple excitation spot for multiphoton fluorescence microscopy. The point spread function (PSF) is generated by interfering two counterpropagating extended depth of focus beams along the optical axis. At an illumination wavelength of 976 nm and aperture angle of 60 degrees , five distinct nanospots of dimension approximately 210 nm is obtained along the optical axis. The resulting PSF has the ability to simultaneously excite multiple planes, and overcomes the sidelobe problem associated with single-photon variant. The proposed multiple-excitation-spot-based-optical imaging technique may find potential application in nanobioimaging and three-dimensional fluorescence microscopy.