Phase spatial light modulators (SLMs) are widely used for generating multifocal three-dimensional (3D) illumination patterns, but these are limited to a field of view constrained by the pixel count or size of the SLM. Further, with two-photon SLM-based excitation, increasing the number of focal spots penalizes the total signal linearly—requiring more laser power than is available or can be tolerated by the sample. Here we analyze and demonstrate a method of using galvanometer mirrors to timesequentially reposition multiple 3D holograms, both extending the field of view and increasing the total time-averaged two-photon signal. We apply our approach to 3D two-photon in vivo neuronal calcium imaging using a field microscope (LFM) http:clarityresourcecenter.com/functional3D.html to enable single-snapshot 3D visualization of the excited fluorescence.

Extended field-of-view and increased-signal 3D holographic illumination with time-division multiplexing. Yang SJ, Allen WE, Kauvar I, Andalman AS, Young NP, Kim CK, Marshel JH, Wetzstein G and K Deisseroth. Optics Express 23(25):32573–32581.