Simultaneous multilayer scanning and detection for multiphoton fluorescence microscopy

Fast three-dimensional (3D) imaging requires parallel optical slicing of a specimen with an efficient detection scheme. The generation of multiple localized dot-like excitation structures solves the problem of simultaneous slicing multiple specimen layers, but an efficient detection scheme is necessary. Confocal theta detection (detection at 90 degrees to the optical axis) provides a suitable detection platform that is capable of cross-talk-free fluorescence detection from each nanodot (axial dimension approximate to 150 nm). Additionally, this technique has the unique feature of imaging a specimen at a large working distance with super-resolution capabilities. Polarization studies show distinct field structures for fixed and fluid samples, indicating a non-negligible field-dipole interaction. The realization of the proposed imaging technique will advance and diversify multiphoton fluorescence microscopy for numerous applications in nanobioimaging and optical engineering.