3-photon excitation enables in vivo fluorescence microscopy deep in densely labeled and highly scattering samples. To date, 3-photon excitation has been restricted to scanning a single focus, limiting the speed of volume acquisition. Here, for the first time to our knowledge, we implemented and characterized dual-plane 3-photon microscopy with temporal multiplexing and remote focusing, and performed simultaneous in vivo calcium imaging of two planes beyond 600 µm deep in the cortex of a pan-excitatory GCaMP6s transgenic mouse with a per-plane framerate of 7 Hz and an effective 2 MHz laser repetition rate. This method is a straightforward and generalizable modification to single-focus 3PE systems, doubling the rate of volume (column) imaging with off-the-shelf components and minimal technical constraints.
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