Figure 2. Implementation of near-simultaneous two-photon and three-photon excitation. A, Schematic of the optical layout for near-simultaneous two-photon and three-photon excitation; 1300-nm beam (black) passed a Pockels cell (PC), prism compressor, a collimating telescope, combining dichroic mirror (CD), x-y galvanometer pair (G), scan lens (SL), tube lens (TL), FF735-DI02 primary dichroic mirror (PD), and objective lens; 910-nm beam (red) passed a PC, beam expansion to ∼1 cm in diameter, electrically-tunable lens (ETL), 0.3× beam expansion before being reflected by the combining dichroic mirror onto the galvanometer pair. B, Scanning strategy for near-simultaneous two-photon and three-photon excitation. Red: 920-nm excitation, no 1300-nm excitation. Black: no 920-nm excitation, 1300-nm excitation. Gray: both lasers blocked. Lines were sorted into two-photon and three-photon images. C, Example images from 350 µm below the pia, with traces from two somata.