Simultaneous two-plane, two-photon imaging based on spatial multiplexing

The two-photon microscope is a powerful tool in life science. Conventional two-photon microscopy can image only a plane of a particular axial position at a time. Axial scanning can get the volumetric information, but it gets signals from different axial positions serially, which means that the exposure time at every plane is limited. Here we demonstrate a novel method, to the best of our knowledge, that can simultaneously record images from two planes at different xyz positions. The demultiplexing of the signal is realized using a confocal strategy. The experimental results show that it can be used for simultaneous two-photon imaging at two focal planes with little cross talk. (C) 2018 Optical Society of America